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Functional analysis of arabidopsis general transcription factors TBP and TFIIB

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Functional analysis of arabidopsis general transcription factors TBP and TFIIB
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14-3-3 Proteins ( jstor )
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DNA ( jstor )
Genes ( jstor )
Genetic mutation ( jstor )
In vitro fertilization ( jstor )
Proteins ( jstor )
RNA ( jstor )
Stirrups ( jstor )
Yeasts ( jstor )
Arabidopsis -- Genetics ( lcsh )
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Thesis (Ph.D.)--University of Florida, 1999.
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Includes bibliographical references (leaves 121-142).
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by Songqin Pan.

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FUNCTIONAL ANALYSIS OF ARABIDOPSIS GENERAL
TRANSCRIPTION FACTORS TBP AND TFIIB













By

SONGQIN PAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1999



























In memory of my father, Guoxiang Pan














ACKNOWLEDGEMENTS


I am deeply grateful to my mentor, Dr. Bill Gurley, for his valuable professional

advice and personal support, which have made this work and my future career possible.

I also thank Dr. Rob Ferl for his collaboration in this study and valuable advice as a

committee member. Thanks are extended to Drs. Ken Cline, Don McCarty, and James

Preston III for their expertise, lab resources, and valuable advice as committee members;

to Drs. Curt Hannah and Charles Guy for their lab resources; to Dr. Prem Chourey for

the maize cell line; to present and past members of the Gurley lab: Drs. Eva Czarnecka-

Verner, Don Baldwin and Eloise Adams for their valuable clones and discussion; to the

DNA Sequencing Core of Microbiology and Cell Science Department, University of

Florida, for confirming the mutation constructs; to the students, staff, and faculty of

PMCB Program and Microbiology and Cell Science Department for their friendship.

With deep gratitude I thank my family for their support and understanding throughout

the course of this study.














TABLE OF CONTENTS




ACKNOWLEDGEMENTS ............................................................................... ...... iii

LIST OF ABBREVIATIONS ................................................................................ vii

AB STRA CT ....................................................................................... ...................... ix

CHAPTERS

1 LITERATURE REVIEW...............................................................................

The Pre-Initiation Complex.......................................................................... 1
T FIID ...................................................................................................................5
The Holoenzyme .............................................................................................9
General Transcriptional Factor TBP .............................................................. 13
General Transcriptional Factor TFIIB.......................................... ............. 19
TBP-TFIIB Interaction....................................................... ....................23
Interactions of TBP and TFIIB with Transcriptional Regulatory Proteins ...........26
Interactions with Activators........................ .......................................26
Interactions with repressors .............................................. ..................... 31
Transacting Proteins...................................................................................32
GAL4 ...................................................................................................... 32
V P16............................................... ...................................................... 34
Ftz...................................................... ....................................................35
V P ............................................... ......................................................... 36
LpH SF8................................................................................. .................37
The 14-3-3 Proteins ..................................................................................37
Rationale for the Present Study.............................. .........................................38

2 CRITICAL ROLE OF THE TBP-TFIIB INTERACTION IN SUPPORTING
ACTIVATED TRANSCRIPTION IN PLANT CELLS................................39

Introduction................................................................................................. 39
Materials and Methods ..................................................................................46
Mutagenesis for AtTBP2 ..........................................................................46
Particle Bombardment in Maize Suspension Cells.........................................47









GUS and Luciferase Assays......................................................................51
Protein Expression and Purification from E. coli ...........................................51
In vitro Protein Translation.........................................................................52
In vitro GST Pull-Down Assay....................................................................52
R results .............................. .............. ........................................................... 53
The C-terminal Stirrup of AtTBP2 is Required for Binding to AtTFIIB.........53
The TBP-TFIIB Interaction is Dispensable in Basal Transcription in vivo .....55
The TBP-TFIIB Interaction is Required for Activated Transcription by
the GAL4 Prom other ............................................................................. 59
Requirement for TBP-TFIIB Interaction Confirmed Using an Altered
Specificity TBP ................................................................................ 67
Complex Promoters Show Much Less Dependence on the TBP-TFIIB
Interaction ........................................................................................... 73
Reliance on Multiple Activation Pathways Can Partially Compensate
Suppression by TBP Stirrup Mutations .............................................77
D iscussion.................................................................................................... 79

3 THE SPECIFIC INTERACTIONS WITH TBP AND TFIIB IN VITRO
SUGGEST 14-3-3 PROTEINS MAY PARTICIPATE IN THE
REGULATION OF TRANSCRIPTION WHEN PART OF A DNA
BINDING COMPLEX ..............................................................................88

Introduction.................................................................................................. 88
Materials and Methods ...............................................................................92
Protein Expression in E. coli ......................................................................92
Protein Purification from E. coli Lysate ................................................93
In vitro Protein Translation ......................................................................94
In vitro GST Pull-Down Assay ..................................................................94
Site-Specific Point Mutagenesis ................................................................95
Transient Expression Assay .......................................................................95
Results ......................................................................................................... 96
Plant 14-3-3 Proteins Interact with Human TFIIB in vitro ..........................96
Conserved C-terminal Core of hTFIIB Binds Arabidopsis 14-3-3 Protein......98
Arabidopsis 14-3-3 Protein and VP16 Show Similarities in Interactions
w ith hTFIIB .......................................................................................... 100
Human 14-3-3 u Shows Affinity for Human TFIIB, TBP, and TAFn32,
but not for TAF155................................................................................. 104
Human 14-3-3 u Contains Two Domains That Bind TFIIB.......................... 105
Alanine Substitutions in 14-3-3 Helix 7 Identify Amino Acids Critical
for Binding TBP and TFIIB .................................................................105
At 14-3-3 < Stimulates GAL4/GUS Expression in Onion Cells.................. 108
D iscussion............................................ .............................. .......... ............ 114









LIST OF REFEREN CES .............................................................................................121

BIO GRAPH ICAL SKETCH ..................................................................................... 143














LIST OF ABBREVIATIONS


AD ..................................................................................................... activation domain
AdM L ...........................................................................adenovirus major late promoter
bp .................................................................................................. .................... base pair
CaM V .................................................................................. cauliflower mosaic virus
Cpm ............................................................................................... counts per minute
CTD ......................................................................................... carboxy-term inal domain
DAB..................................................... a complex of TATA, TBP, TFIIA and TFIIB
DB.................................................................... a complex of TATA, TBP, and TFIIB
DBD .............................................................................................. DNA-binding domain
EM SA .......................................... .................... electrophoretic mobility shift assay
Ftz..................................................... ................................................... fushi taratzu
GST........................................................................................ glutathione S-transferase
GUS/LUC...................................................... relative P-glucuronidase/luciferase activity
HeLa...................................................................................... a human cancer cell line
hr........... ....................................... .......... ........................................ ......... hour
HTH ..................................................................................................... helix-turn-helix
LpHSF8.............................................................. tomato heat shock transcription factor 8
min....................................................................................................................... m minute
m l...................... .................................................................................. ...............mililiter
mM ............... .......................................... .................................................. m ilimolar
NM R .................................................................................... nuclear magnetic resonance
PCR.......................................................................................polymerase chain reaction
PIC .......................................................................................... the pre-initiation complex
Pol II ............................................................................................RNA polymerase II
RAP30.................................................................................... the small subunit of TFIIF
RAP74................................................................................ the large subunit of TFIIF
sec ........................................................................................................................ second
SL1......................................................... TBP-containing factor for RNA polymerase I
SRB ........................................................... suppressor of CTD mutations of RNA pol II
TAF............................................................................................. TBP-associated factor
TATA ...................................................................................... core promoter element
TBP .............................................................................................. TATA-binding protein
TFIIA ...... .. .......... ......................................................... transcription factor for pol II A
TFIIB ............................................................................ transcription factor for pol II B
TFIID ............................................................................ transcription factor for pol II D
TFIIE.................................................................. transcription factor for pol II E
TFIIF................................................................................transcription factor for pol II F
TFIIH ......................................................................... transcription factor for pol II H









TFIIIB ................................................. TBP-containing factor for RNA polymerase III
TGTA .............................................................................................. ...... m utated TATA
TRF ............................................................................................... TBP-related factor
g........................................... ................................................................... m icrogram
.............................................................................................................. ........ m icroliter
VP1 .................................................................................................. viviparous protein 1
VP16 ..............................................................................herpes simplex virus protein 16












































Viii













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

FUNCTIONAL ANALYSIS OF ARABIDOPSIS GENERAL
TRANSCRIPTION FACTORS TBP AND TFIIB

By

Songqin Pan

May 1999


Chairman: William B. Gurley, Ph.D.
Major Department: Plant Molecular and Cellular Biology Program

Studies with human, Drosophila and yeast systems have demonstrated that the

general transcriptional factors, TBP and TFIIB, play crucial roles in the transcription of

class II genes in eukaryotes. These two proteins not only are important in assembly of

the pre-initiation complex (PIC), but also serve as critical targets for recruitment by

activation domains in activated transcription. To evaluate the role of TBP-TFIIB

interaction in transcription, a series of mutations in the C-terminal stirrup of arabidopsis

TBP2 designed to disrupt TBP-TFIIB interaction were introduced into plant cells

(maize) using transient assays. TBP-TFIIB interaction was shown to be dispensable for

basal transcription, but required for activated transcription driven by different types of

activation domains in the GAL4-dependent transcription system. Inhibition of

transcription by TBP mutation ranged from 5- to 16-fold indicating that association of

TBP and TFIIB varied in importance depending on the activation domains. In vitro

protein binding experiments showed that both E146 and E144 of AtTBP2 are essential

ix








for TFIIB binding, however, mutation E146R showed much more severe inhibition on

activated transcription than E144R in vivo. The natural CaMV 35S and maize ubiquitin

promoters showed independence of TBP-TFIIB interaction for their activities, indicating

that TBP-TFIIB interaction is not required for transcription driven by complex

promoters.

In a second series of experiments, the potential of 14-3-3 proteins to regulate

transcription in plants was evaluated by a combination of in vitro and in vivo studies. In

vitro, 14-3-3 proteins have specific affinity for both TBP and TFIIB as demonstrated by

domain mapping and alanine substitution analysis. 14-3-3 and VP16 seemed to share the

same or overlapping binding domain within TFIIB, and competed with each other in the

same TFIIB binding reaction. Given the fact that 14-3-3 interacts with several activators

such as VPI, EmBPI and glucocorticoid receptor, 14-3-3 proteins may have role(s) as

transcriptional co-activators to bridge the activators and the PIC. However, the

Gal4DBD/14-3-3 chimera showed a low or no activation potential in plant cells,

probably due to low protein level of the Gal4DBD/14-3-3 with appropriate function to

recognize the GAL4 binding sites.














CHAPTER 1
LITERATURE REVIEW

The Pre-Initiation Complex


The protein-coding genes in eukaryotic cells are transcribed by RNA polymerase

II, which is a multi-subunit enzyme consisting of at least ten subunits in yeast (Woychik

and Young, 1990). Within the total enzyme complex, only the largest subunit RPB1 has

the catalytic activity. The C-terminal domain (CTD) of RPB1 is the regulatory domain

of the enzyme. The CTD contains a peculiar sequence of as many as 26 YSPTSPS

heptapeptide repeats in yeast (Lee and Greenleaf 1989), 40 repeats in arabidopsis

(Dietrich et al., 1990) and 52 repeats in mouse (Corden et al., 1985). These repeated

sequences could be hypo- or hyper-phosphorylated at their serine and threonine residues

(Cadena and Dahmus, 1987). The phosphorylation/dephosphorylation of the CTD can

regulate transcriptional activity of RNA polymerase II. The enzyme is in a

transcriptionally active state when it is phosphorylated (Cadena and Dahmus, 1987),

while the dephosphorylation can arrest transcription (Dubois et al., 1994). However,

despite the inherent transcriptional function, RNA polymerase II alone is not sufficient

for gene-specific transcription because its highly nonspecific affinity with DNA results

in random transcription (Weil et al., 1979). For the proper transcription of a given gene,

the enzyme requires a group of proteins, the general transcriptional factors, to form the

pre-initiation complex (PIC) to specifically recognize the core promoter of the gene and

correctly initiate transcription (Kollmar and Farnham, 1993). The assembly of the PIC

I






2

on the core promoter can be facilitated by a transcriptional activator or inhibited by a

transcriptional repressor, which results in transcriptional activation or repression

respectively.

Initial biochemical characterization studies of several in vitro transcription

systems suggest that many general transcription factors together with RNA polymerase

II assemble on the promoter of a DNA template in a stepwise fashion in order for

transcription to occur (Weinmann, 1992). These experiments were primarily based on

the chromatographic purification of nuclear proteins. Using various ionic exchange

columns, RNA polymerase II and several general transcription factors were purified

from the nuclear extracts. Their designations were initially determined by their order of

elution and are as follows: TFIID (Matsui et al., 1980), TFIIA (Cortes et al., 1990;

Samuels and Sharp, 1986), TFIIB (Ha et al., 1991; Matsui et al., 1980), TFIIE (Flores et

al., 1989; Inostroza et al., 1991), TFIIF (Flores et al., 1990) and TFIIH (Flores et al.,

1992). By sequential addition of the purified enzyme and factor proteins, in vitro PIC

assembly can be accessed. However, the high order PIC complex is formed only when

these proteins are added into the reaction in the fixed orders (Flores et al., 1992). These

observations led to the hypothesis that formation of a functional PIC involves the

multistep addition of general transcription factors in an ordered sequence.

According to the multistep hypothesis based on in vitro transcription systems in

both HeLa and yeast, the TATA binding protein (TBP) ofTFIID is the first protein to

recognize the DNA TATA motif of the core promoter (Buratowski et al., 1989;

Horikoshi et al., 1989; Maldonado et al., 1990). Following TBP binding to TATA,

TFIIA and TFIIB interact with both TBP and DNA to form DAB complex (Buratowski








et al., 1989; Maldonado et al., 1990). This complex now functions as a platform for

docking of the polymerase to the promoter. This process is accomplished by three

interactions: TBP with CTD of the largest subunit of polymerase (Usheva et al., 1992),

the bridge of the RAP30 subunit of TFIIF between polymerase and TFIIB involving its

zinc-finger domain (Ha et al., 1993) and a direct interaction of polymerase with the

second direct repeat of TFIIB C-terminal core (Ha et al., 1993). As the final steps in PIC

assembly, TFIIE and TFIIH subsequently enter the complex to result in formation of a

complete functional PIC (Flores et al., 1992). In the form of the PIC, RNA polymerase

II is able to initiate RNA synthesis from a specific starting site with the aid of the

helicase activity of TFIIH to melt the DNA template in the presence of ATP (Holstege et

al., 1996).

Recently, a new approach has been taken to directly probe the structure of the

pre-initiation complex in vivo. The immunoprecipitation of nuclear extracts using

antibodies against either a general transcription factor or a subunit of the polymerase II

identified a multi-protein complex, the RNA polymerase II holoenzyme (Kim et al.,

1994b; Ossipow et al., 1995). The holoenzyme complex contains RNA polymerase II,

most general transcription factors and mediator proteins like SRBs. However, TFIID is

not included in holoenzyme (Kim et al., 1994b; Ossipow et al., 1995). This finding

suggest that in contrast to in vitro stepwise assembly, in vivo most of the general

transcription factors are pre-associated with RNA polymerase before assembly on the

promoter. Therefore, in vivo the PIC assembly is likely much simpler than that in vitro.

The whole process probably involves only two steps directly: the recognition of the core






4

promoter by TFIID and the association of TFIID with the holoenzyme mediated by the

interactions between their respective components.

From the finding of the holoenzyme, one would expect that the recruitment of

any components of the two megacomplexes, TFIID and holoenzyme, to the promoter to

correctly assemble the PIC should be able to activate transcription. In contrast, the

stepwise model predicts that only the recruitment of TFIID, which is involved in the

very first step of the assembly pathway of the PIC, would most significantly stimulate

transcription. This hypothesis has been tested by several labs with the approach of

artificial recruitment in which a component of either TFID or holoenzyme is directly

fused to a DNA binding domain (DBD). The tested components include TBP (Chatterjee

and Struhl, 1995; Klages and Strubin, 1995; Xiao et al., 1997; Xiao et al., 1995b),

TAF1is (Gonzalez-Couto et al., 1997), TFIIB (Gonzalez-Couto et al., 1997; Lee and

Struhl, 1997) and mediator proteins (Barberis et al., 1995; Gaudreau et al., 1997). All of

them show strong stimulation of transcription when tethered to a DNA binding element,

and the level of stimulation is comparable to that obtained by a typical activation

domain such as VP16 (Gonzalez-Couto et al., 1997). These experiments have clarified

that formation of the PIC does not require stepwise recruitments in vivo. Instead,

recruitment of a single component of either the holoenzyme or TFIID can lead to

assembly of a functional pre-initiation complex on the promoter. Recruitment of first

complex, either TFIID or the holoenzyme, presumably results in subsequent recruitment

of the second, which may be mediated by many protein-protein interactions between

their components as characterized in vitro (Goodrich et al., 1993; Ha et al., 1993;

Usheva et al., 1992).






5

TFIID


TFIID is a complex comprised ofTBP (TATA binding protein) and TAF,,s (TBP

associated factors). TBP is the only protein commonly required for transcription by all

three RNA polymerases in eukaryotic organisms (Cormack and Struhl, 1992). Although

TBP by its own is able to bind the TATA element of the core promoters in vitro, the

specificity of promoter recognition by TBP in vivo is likely determined by TAF proteins.

Distinct TAFs are associated with TBP on the core promoters of different classes of

genes to form SL1 (Comai et al., 1992), TFIID (Timmers et al., 1992) and TFIIIB

(Taggart et al., 1992) complexes to support transcription by Pol I, Pol II and Pol III,

respectively. These TAFs may serve as promoter selectivity factors for different classes

of genes (Jordan et al., 1996; Taggart et al., 1992; Verrijzer et al., 1995), or may

facilitate the recognition by the corresponding polymerase by interactions with its

component proteins, or may provide the targets for the upstream activator proteins in

activated transcription (Beckmann et al., 1995).

The TFIID complex is believed to have several forms. The basic, or general,

form of TFIID presumably functions in all polymerase II transcription, and contains at

least eight different TAFs (Burley and Roeder, 1996). cDNAs for TAF,,s have been

isolated from human, yeast and Drosophila with predicted protein sizes ranging from

15-250 kD (Burley and Roeder, 1996). In vitro, the proteins expressed by these cDNAs

together with TBP are able to reconstitute the TFIID complex (Chen et al., 1994).

Although there has not been a TAF,, identified from plants, it has been shown that there

is a TFIID activity in plant cells (Washburn et al., 1997), and several expressed

sequence tags (EST) clones from maize and arabidopsis show sequence similarity to








known TAF,,s proteins in animals and yeast. A second form of TFIID appears to be

tissue-specific, and is found associated with specific cell types in Drosophila (Hansen et

al., 1997). The tissue-specific TBP and TAF,,s in this complex are distinct from those

ubiquitous ones in the general TFIID and are named TRF and nTAFs respectively

(Hansen et al., 1997). Finally, there is another TFIID-like complex that lacks TBP

(Wieczorek et al., 1998). The complex contains several TAF, s and other unknown

proteins. This TBP-less complex also has the normal function of TFIID in transcription

of both TATA-containing and TATA-less promoters in vitro, but the implication in

terms of physiological function is unclear.

For the general TFIID, its architecture appears to be highly conserved among

eukaryotic organisms. The largest TAF,, (hTAF,,250, dTAF,,250 and yTAF,130) is

thought to serve as a primary anchor to TBP; the two proteins together act as a platform

for the entry of other TAF,,s into the TFIID complex by the interactions with either TBP

or the largest TAF. (Burley and Roeder, 1996). For instance, in the assembly of

Drosophila TFIID, TBP interacts with TAF,,s 150 (Verrijzer et al., 1994), 80 (Kokubo et

al., 1993b), 62 (Kokubo et al., 1994; Weinzierl et al., 1993b), 42 (Kokubo et al., 1994)

and 30a/22 (Kokubo et al., 1994; Yokomori et al., 1993). dTAF,250 interacts with

dTAF,,s 110 (Kokubo et al., 1993a; Weinzierl et al., 1993a), 150 (Verrijzer et al., 1994),

62 (Weinzierl et al., 1993b) and 303 (Yokomori et al., 1993). The small TAF,s also

participate in interactions with each other to further stabilize the TFIID complex

(Kokubo et al., 1994; Kokubo et al., 1993b; Weinzierl et al., 1993b; Yokomori et al.,

1993). However, there is an important caveat in that these interactions are all








characterized by in vitro biochemical approaches. The importance of each individual

interaction for TFIID function in living cells remains to be demonstrated.

Functional analysis of the TFIID complex reveals distinct requirements for

TAF1s in both basal and activated transcription. In the Drosophila in vitro transcription

system, the TBP/TAF,250 binary complex is sufficient to support the basal transcription,

but not activated transcription, which requires additional TAF, proteins depending on

the specific transactivator bound to the promoter (Chen et al., 1994). For example,

dTAF, 150 and dTAF, 10 are required for transcription activated by NTF-1 and Spl,

respectively (Chen et al., 1994). In these systems, dTAFns are believed to play specific

co-activator roles (Chen et al., 1994). The importance of TAFns in activated

transcription is further confirmed in vivo where dTAFns 60 and 100 mutants failed to

support transcription of bicoid-dependent target genes during Drosophila embryo

development (Sauer et al., 1996). In contrast to a clear dependence on TAFns for

activated transcription in metazoans, deletion or inactivation of several TAF, genes in

yeast does not affect transcription by several activator proteins, indicating that TAF,,s are

not generally required in activated transcription in yeast (Moqtaderi et al., 1996; Walker

et al., 1996). Although, apparently, there is a functional TFIID complex in yeast cells

(Gonzalez-Couto et al., 1997), it is likely that not all TAFs are generally required in

activated transcription, but, rather, they are selectively used in an activator- or promoter-

dependent manner. Even in animals, the co-activator function of TAFus seems to be

redundant with other general co-activator proteins like PC4 (positive co-activator 4) and

pX which are able to support transcription in the absence of TAFns in the human in vitro

transcription systems (Haviv et al., 1996; Wu et al., 1998).








In addition to co-activator roles, some TAFs also have affinity for DNA and

enzymatic activity as well. For the TATA-less core promoter of class II genes, the TBP

protein is unable to specifically bind to DNA. This problem can be overcome by the

initiator element which is located around the transcription start site. This element can be

recognized by some TAF,, proteins that seem to tether the TFIID complex at the core

promoter allowing for transcription (Martinez et al., 1994). Interestingly, some TAFs

appear to have enzymatic functions as seen with TAF,250 which has both kinase and

acetyltransferase activities (Dikstein et al., 1996; Mizzen et al., 1996). The protein alone

or in TFIID is able to phosphorylate the RAP74 subunit of TFIIF at its serine residues

(Dikstein et al., 1996). The phosphorylation is done by the cooperative action of the two

kinase domains of TAF,250, with each capable ofautophosphorylation. It is thought that

this phosphorylation may provide a signal within the PIC for transcriptional regulation

(Dikstein et al., 1996). On the other hand, the histone acetyltransferase activity of

TAF,250 (Mizzen et al., 1996) may be important in dissociating the histone core from

DNA to facilitate nucleosome remodeling, since the acetylation of histone proteins

inhibits histone-DNA interactions (Mizzen et al., 1996; Puig et al., 1998). Another

example is seen with human TAF,170 which was shown to have an ATPase activity

(Chicca et al., 1998). hTAF,170 binds to the TBP core region in the TBP-DNA complex

and hydrolyses ATP. The generated energy contributes to the dissociation of TBP from

DNA and TAF1,170 from TBP as well (Chicca et al., 1998). As expected, transcription is

inhibited by this ATPase activity when TBP is used in the reaction. However, either

TFIID or TFIIA included in the transcriptional reaction appears to be able to reverse the

TAFi70-mediated repression (Chicca et al., 1998).






9

Another striking feature of TFIID is that some TAF,s are homologs ofhistone

proteins. dTAFA2 and hTAF,31 are homologous to H3; dTAF,,62 and hTAF,,80 are

homologous to H4; whereas, dTAF,,30o/22 and hTAF,20/15 are putative H2B homologs

(Burley and Roeder, 1996). H2A homologs, however, are still lacking. The fact that

some TAF,s are histone-like proteins suggests that TAF, proteins may play role(s) in

nucleosome remodeling to facilitate TFIID binding to the core promoter. This possibility

is strongly supported by the cocrystal structure of two Drosophila TAF,, proteins, in

which the dTAFA2/dTAF,k62 heterotetramer very much resembles the histone octamer

core structure (Xie et al., 1996). This mimic to the histone core may allow TFIID to

compete with true histones for binding to a chromosomal promoter.

The Holoenzyme


The eukaryotes' RNA polymerase II is believed to exist in cells in a complex

with its many associated factor proteins (Kim et al., 1994; Ossipow et al., 1995; Wilson

et al., 1996). This holoenzyme complex is the functional form of RNA polymerase II

and probably contains all activities necessary for a chromosomal gene transcription

except that it lacks the TATA-box binding function. The holoenzyme probably has

several forms, since its composition appears to be different from one prep to another

(Cho et al., 1997; Ossipow et al., 1995; Scully et al., 1997). In general, TFIIB is

considered part of the holoenzyme, but TFIID exists as a separate complex before

forming the PIC with holoenzyme on the core promoter. This was tested by the mutation

of TATA motif, TGTA, to distinguish whether activation of transcription is through

recruitment of TFIID or the holoenzyme. Direct tethering of a TFIID component, a TAF

protein, to the promoter, for example, can artificially localize TFIID complex to the








promoter, and transcription should not be very sensitive to the TGTA mutation. In

contrast, transcription by direct tethering of a holoenzyme component to the promoter,

for example, TFIIB, can occur with only TATA but not TGTA motif, because of lack of

stable binding of TFIID to the TGTA. A study conducted in yeast cells has proven that

this is the case, demonstrating that in vivo, the TFIB-containing complex, presumably

the holoenzyme, and the TAF-containing complex, presumably TFIID, are separate

before joining to the promoter (Gonzalez-Couto et al., 1997).

Functional analysis has shown that the holoenzyme is able to respond to

activators to stimulate transcription (Koleske and Young, 1994). The purified yeast

holoenzyme is required for transcription in vitro activated by the GAL4-VP16 activator,

while the purified RNA polymerase and general transcription factors fail to respond to

the same activator, suggesting that holoenzyme is the functional form of RNA

polymerase II (Koleske and Young, 1994). This holoenzyme-dependent transcription is

thought to occur through recruitment of the holoenzyme by the activator protein to

increase the local concentration of the enzyme on the promoter high enough for its

function. Consistent with this idea, with a low holoenzyme concentration an activator is

required for high level of transcription, but the same level of transcription can be

achieved without an activator when high holoenzyme concentration is used in the in

vitro transcription reaction (Gaudreau et al., 1998). An activator does not further

stimulate transcription when high holoenzyme concentration is used (Gaudreau et al.,

1998). These findings suggest that an activator stimulates transcription by simple

recruitment of the holoenzyme to the promoter without altering the properties of the

complex.








The mediator fraction of the holoenzyme is tightly associated with the CTD

domain ofpolymerase II and consists of some twenty polypeptides including SWI/SNF

(Wilson et al., 1996) and SRB (suppressor of RNA polymerase CTD deletion mutants)

proteins (Chao et al., 1996; Kim et al., 1994b). The SWI/SNF proteins have important

functions in remodeling the chromatin structure. These proteins are able to facilitate

binding of both an activator protein and the PIC components to the promoter DNA in

vitro by disrupting the nucleosomes (Cote et al., 1994; Imbalzano et al., 1994a).

Consistent with their roles in nucleosome disruption, the SWI/SNF proteins were shown

to have high DNA binding affinity and ATPase activity (Quinn et al., 1996; Richmond

and Peterson, 1996). The properties of DNA binding activity are similar to those

exhibited by HMG-box containing proteins. The complex recognizes the minor groove

of the DNA helix and is able to interact with the synthetic four-way junction DNA

(Quinn et al., 1996). Nucleosome disruption is an ATP-dependent process. The ATPase

domains of the SWI/SNF proteins hydrolyze ATP when stimulated by DNA binding.

This ATPase activity appears to be critical to SWF/SNF function, because point

mutations introduced into the ATPase domains resulted in a dominant negative

phenotype in yeast cells (Richmond and Peterson, 1996). The functional importance of

chromatin remodeling by SWI/SNF in transcriptional activation has been shown by

several studies in which a nucleosomal promoter became more accessible to both

upstream activators and PIC components in the presence of SWI/SNF proteins to result

in high levels of transcription in vivo (Burns and Peterson, 1997; Ryan et al., 1998; Wu

and Winston, 1997). Therefore, association of the SWI/SNF proteins with the








polymerase is believed to enable the enzyme to overcome transcriptional repression by

chromosome structure.

The SRB proteins of the mediator complex appear to play both positive and

negative roles in transcription. With temperature-sensitive mutations in the SRB genes,

yeast cells show a rapid and general shutdown of mRNA synthesis when transferred to

the restrictive temperature, suggesting that some SRBs are required for transcriptional

activation (Thompson and Young, 1995). The positive effects by the SRBs may be

related to their roles in serving as the targets for activator proteins. The study by Koh et

al. (1998) has clearly shown that in vivo Srb4 enhances transcription by Gal4 AD

through direct interaction between the two proteins, since reciprocal mutations in both

proteins can restore transcriptional activity. Srb4 may serve as a general target for many

types of activation domains, because it is an essential factor for expression of most class

II genes in yeast (Lee et al., 1998). On the other hand, several other Srbs exert a

negative influence on transcription. The human NAT complex contains Srb7, SrblO and

Srbl 1, and is a negative regulator of activated transcription (Sun et al., 1998). It

associates with the human RNA polymerase II holoenzyme and phosphorylates the CTD

domain at residues distinguished from those by the kinase of TFIIH (Sun et al., 1998).

However, it is unknown how this activity can negatively regulate transcription. In some

cases, the negative SRBs can function by serving as the targets for the repressors. For

example, the yeast repressor Sfll represses the SUC2 gene by recognizing the repression

site located immediately 5' to the TATA element and directly targeting Srb 9 and Srbl 1

(Song and Carlson, 1998). Presumably, the interactions of Sfll with these SRB proteins






13

prevent formation of a functional PIC complex on the promoter. Overall, it seems that

distinct sets of SRBs are used in either transcriptional activation or repression.

General Transcription Factor TBP


The central component of the TFIID complex, TBP, is the first general

transcription factor cloned and characterized. The cDNAs for TBP have been isolated

from many eukaryotic organisms, including animals (Kao et al., 1990; Muhich et al.,

1990), plants (Gasch et al., 1990; Haass and Feix, 1992) and yeast (Horikoshi et al.,

1989). While the TBP gene appears to be single copy in both animals and yeast, there

are at least two copies in plants, including both dicots (arabidopsis) (Gasch et al., 1990)

and monocots (maize) (Haass and Feix, 1992). The known TBP protein sequences show

a high degree of conservation in their C-terminal 180 amino acids, which is the

functional core domain for binding the TATA motif and supporting transcription (Hoey

et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990). In contrast, the N-terminal

region of the proteins is much diverged, and its function is not clear. Deletion of this

region does not affect polymerase II transcription and yeast cell growth, indicating that

it is not important in TBP function. However, inclusion of the antibody mAblC2 against

this region inhibits in vitro transcription from TATA-containing, but not TATA-less

promoters (Lescure et al., 1994). Another study indicates that the N-terminal region of

hTBP down-regulates the binding of TBP with the U6 TATA box of the polymerase III

promoter (Mittal and Hernandez, 1997). These results suggest that the N-terminal region

may have a regulatory role on TBP function. Recently, a tissue-specific TBP homolog,

TRF, was identified from Drosophila (Hansen et al., 1997). Like TBP, TRF possesses

normal function in TATA binding, DB complex formation, and supports both basal and






14

activated transcription (Hansen et al., 1997). This finding suggests that a homolog of

TBP is required for the cell-type-dependent transcription in addition to its general roles

in transcription.

The protein structure of the arabidopsis TBP isoform 2 has been revealed by X-

ray crystallography studies (Kim and Burley, 1994; Nikolov and Burley, 1994). AtTBP2

is the smallest TBP protein known, and contains only 200 amino acid residues. The total

molecule is almost just the core domain with an N-terminal loop of only eighteen amino

acids (Gasch et al., 1990). In protein crystals, AtTBP2 is organized as two structural

repeats (Kim and Burley, 1994; Nikolov and Burley, 1994). The structures of the two

repeats are very similar and show strong twofold symmetry. Each repeat contains two a-

helices (almost perpendicular to each other), five anti-parallel p-sheets and one stirrup-

like loop between the second and third p-sheets. The ten p-sheets in total form a

concave surface of the TBP molecule and are responsible for binding to DNA. The

upper convex surface of the molecule is composed of the four a-helices, the basic linker

between the two repeats and the N-terminal loop (Kim and Burley, 1994; Nikolov and

Burley, 1994). This surface is not in contact with DNA and is presumably responsible

for interactions with TAF,,s, holoenzyme components and other transcriptional regulator

proteins.

Interaction with the TATA element of the core promoter is the primary function

of TBP in transcription of class II genes and has been characterized in great detail. The

TBP protein is believed to exist as two forms: monomer and dimer (Coleman et al.,

1995; Taggart and Pugh, 1996). The dimer does not have DNA binding activity and has

to dissociate into the monomer form for binding to DNA (Coleman and Pugh, 1997).








The equilibrium between monomer and dimer forms may be important in regulation of

the TBP-DNA interaction, since this interaction is a slow reaction as demonstrated in

kinetic studies (Coleman and Pugh, 1997). As a monomer, the TBP molecule recognizes

the TATA motif(5'-TATAAAAG-3') at its minor groove when bound to the AdMLP

core promoter (Kim and Burley, 1994). The protein sits astride the DNA with its first

and second direct repeats interacting with the 3' and 5' halves of the eight base pairs of

DNA, respectively. The protein-DNA interaction is largely mediated by hydrophobic

van der Waals contacts and involves an induced-fit mechanism. It is thought that the 5'

half of the TATA element first makes contact with the underside of the second direct

repeat of TBP. As a result of this interaction, both DNA and the protein undergo

significant conformational changes to allow the best fit for the second interaction

between the 3' half of DNA and the first direct repeat of TBP to result in a overall tight

association between the two molecules. The conformational changes in DNA are

characterized as the partially unwound center of the eight base pairs, the widened minor

groove, the severe bend towards the major groove and the severe twist at either end of

the TATA. The insertions ofphenylalanines 148 and 165 into the first base pair and

phenylalanines 57 and 74 into the last base step result in two strong kinks in the DNA

that are believed to be critical in distorting the DNA. As a consequence of protein-DNA

interaction, there also is a significant twist in the relative positions of the two direct

repeats of TBP (Kim and Burley, 1994). In solution, TBP appeared to interact with DNA

in both orientations, with the entries of TFIIA and TFIIB having little effect on the

polarity of the binding (Cox et al., 1997). This result is surprising since one of the

functions of the TATA is thought to involve in determination of polarity with respect to






16

the transcription start site. This controversy in the TBP-DNA interaction between in

crystal and in solution has not been resolved.

There are several interesting features about the TBP-DNA interaction. First,

DNA bending has been proven very important for TBP affinity. When circular, instead

of linear, DNA is used, the TBP-TATA interaction is greatly affected by the location of

the TATA element. TBP binds to TATA very tightly when it is located in a position

where there is a slight bend toward major groove. The binding affinity is 100-fold

greater than binding to an unbent linear TATA (Parvin et al., 1995). Second, TBP seems

to have nonspecific DNA affinity, although it is commonly thought of as the TATA-

specific DNA binding protein. The nonspecific binding of TBP to random DNA

sequences was evidenced by DNase I footprint and detailed kinetic analysis (Coleman

and Pugh, 1995). However, the average affinity of TBP for a random DNA sequence is

about 103 lower than the specific affinity for the TATA motif, and TBP appeared to

translocate along the DNA molecule (Coleman and Pugh, 1995). Therefore, these

authors believed that TBP is able to slide on a DNA template to reach the TATA

element to form stable binding. This model of the TBP-TATA interaction is similar to

that exhibited by the a factor in bacterial transcription (McClure, 1985). Since TBP

contains the amino acid sequences homologous to the a factor (Horikoshi et al., 1989),

it is not surprising to see that they may have a similar mode in interacting with DNA. In

eukaryotes, however, this random scanning of DNA must be strongly directed and

restricted by recruitment mechanisms. Finally, the major groove of the TATA element

seems to also have functional roles in transcription, although it does not interact with

TBP directly. The modification of the major groove by the substitutions of I:C base pair






17

for A:T (TITI) does not affect the binding of TATA with either TBP or TFIID (Lee et

al., 1997). However, this modification significantly inhibits in vitro transcription by

crude HeLa nuclear extracts (Lee et al., 1997). Based on this evidence alone, it is

suggested that the major groove of the TATA element may be also involved in

interactions) with other transcription factors) (Lee et al., 1997).

In addition to class II genes, some tRNA and 5S RNA genes and most promoters

of RNA polymerase III transcribed-genes also contain the TATA element. These TATA

elements can be universally recognized by TBP, which results in transcriptional

initiation by the different polymerases. The question remains that how these TATA-TBP

complexes can be discriminated by the distinct enzymes. One study has shown that the

orientation of the TBP-TATA complex is the key signal distinguishable for polymerase

II and III. Polymerase II uses the "Forward" TATA template to transcribe DNA; in

contrast, polymerase III uses the "Reverse" TATA template (5'-TTTTTATA-3') in the

same transcription reaction (Wang and Stumph, 1995). This conclusion is supported by

the observations that forward transcription is only inhibited by the polymerase II-

specific-inhibitor a-amanitin but not by the polymerase III-specific-inhibitor tagetitoxin,

and transcription from the reverse TATA was inhibited by only tagetitoxin but not a-

amanitin (Wang and Stumph, 1995). These results indicate that the TBP-TATA complex

can function bi-directionally in the same core promoter with the polarity of transcription

determined by the different RNA polymerases.

Besides the important function in recognizing the TATA and association with

TAF,,s, TBP also interacts with several other components of the PIC, including TFIIA

(Geiger et al., 1996; Tan et al., 1996), TFIIB (Ha et al., 1993; Nikolov et al., 1995) and








the CTD of the largest subunit ofpolymerase II (Usheva et al., 1992). These interactions

may be important in facilitating PIC assembly and stabilizing the TBP-TATA complex

as well. In vitro electrophoretic mobility shift assays (EMSA) have shown that TFIIA

and TFIIB can each independently retard the TBP-DNA complex, indicating the

formation of a ternary complex (Imbalzano et al., 1994b). A much greater proportion of

the DNA probes is shifted by each ternary complex than that by the TBP-TATA binary

complex. This observation is interpreted as an indication that TFIIA and TIIB are able

to further strengthen the DNA binding of TBP. In the TATA-TBP-TFIIA-TFIIB

quaternary complex, TFIIA interacts with N-terminal stirrup of TBP on one side, and

TFIIB interacts with the C-terminal stirrup of TBP on the other side. Physically, TBP,

TFIIA and TFIIB together form a "cylindrical clamp" around the double-helix of the

TATA DNA (Lagrange et al., 1996). When either TFIIA or TFIIB binds to TBP in a

complex with the DNA, an extended protection of promoter DNA can be observed by

the DNase I footprinting (Malik et al., 1993; Yokomori et al., 1994). It is believed that

both TFIIA and TFIIB also are able to interact with DNA due to their physical proximity

to DNA when bound to TBP. A UV cross-linking study on the TATA-TBP-TFIIA-

TFIIB quaternary complex has confirmed the DNA binding activity for both TFIIA and

TFIIB (Lagrange et al., 1996). Therefore, the enhanced stability of the association of

TBP with DNA in the presence of TFIIA or TFIIB is likely due, at least in part, to DNA-

TFIIA and DNA-TFIIB interactions. Interaction of TBP with the underphosphorylated

CTD ofpolymerase II may be important in recruiting the holoenzyme to the promoter,

but once assembled, it appears to be inhibitory to promoter clearance for transcriptional

elongation, resulting in paused polymerases on the promoter (Giardina et al., 1992).






19

Phosphorylation of the CTD by a kinase activity of TFIIH is believed to disrupt the

TBP-CTD interaction and to release the polymerase from the promoter to proceed in the

synthesis of RNA (Parada and Roeder, 1996). The functional importance of this step in

transcription has been confirmed by an in vivo study in Drosophila embryonic cells

(Yankulov et al., 1996).

General Transcription Factor TFIIB


TFIIB is another highly conserved general transcription factor among eukaryotic

organisms. The protein in general consists of two domains: the N-terminal domain and

the C-terminal core. The N-terminal domain is composed of about 100 aa residues and

contains the zinc-ribbon and the adjacent highly conserved region. The C-terminal core

is composed of more than 200 aa, and contains two imperfect direct repeats linked by a

small region characteristic of basic amino acids. The structure of the C-terminal core,

instead of the full-length hTFIB, has been characterized by both crystallography

(Nikolov et al., 1995) and NMR in solution (Bagby et al., 1995), because inclusion of

the N-terminal region resulted in structural instability of the protein. The core domain is

a twofold symmetrical molecule as reflected in the positions of two imperfect repeats.

Each repeat consists of five a-helices with an additional helix at the C-terminal end of

the second repeat (Bagby et al., 1995; Nikolov et al., 1995). The a-helices of the first

repeat are designated as Al to El, and their counterparts in the second repeat as A2 to

E2 (Bagby et al., 1995). The El helix is amphipathic with respect to the distribution of

positive residues, and is thought to be a critical domain involved in interactions with

TBP, DNA (Nikolov et al., 1995) and activators such as VP16 (Roberts et al., 1993).

Protease topology analysis on full-length hTFIIB indicates that the N-terminal domain






20

seems to fold onto the core domain through intramolecular interactions to form the

"closed" conformation (Roberts and Green, 1994). TFIIB with this conformation may

exist in a functionally inactive state. The interaction with an activation domain such as

VP16 can disrupt the intramolecular interaction of TFIIB to induce the "open"

conformation that is believed to be in the functionally active state (Roberts and Green,

1994). However, a word of caution must be interjected with respect to the biological

significance of the closed and open states since these studies were conducted in vitro

and TFIIB seems to function in vivo only when associated with the holoenzyme.

TFIIB is a crucial protein in polymerase II-dependent transcription. The deletion

analysis ofhTFIIB reveals that almost the entire molecule is required for basal

transcription in vitro (Ha et al., 1993; Hisatake et al., 1993). This seems reasonable

since both the N-terminal domain and the C-terminal core have important roles in the

assembly of the PIC (Ha et al., 1993). The zinc-ribbon in the N-terminal domain of

TFIIB is responsible for the interaction with the small subunit RAP30 of TFIIF

(Buratowski et al., 1989; Ha et al., 1993). The binding of TFIIB to TFIIF seems likely to

be critical in joining TFIIB to the holoenzyme. The single point-mutation C37S in this

domain can disrupt the interaction with RAP30 (Buratowski et al., 1989). For the

adjacent conserved region, one known function is to determine the transcription start-

site. Mutations in this region at E62K, R78C, or E62K/R78C, in yeast sua7 (the TFIIB

gene) showed a downstream shift of the start-site for transcription of the cycl gene

(Pinto et al., 1994). This mutant phenotype can be reversed by a second mutation in the

large subunit RAP74 of TFIIF (SSU71 gene) which restores the normal pattern of

transcriptional initiation of the cycl gene (Sun and Hampsey, 1995). This result suggests








that there may be a functional interaction between RAP74 and the conserved region of

TFIIB (Sun and Hampsey, 1995). However, the corresponding mutations in arabidopsis

TFIIB E43R did not show a shift in the start-site of transcription using a hTFIIB-

depleted HeLa nuclear extract in vitro (Baldwin, 1997). It is possible that the residues

involved in start site selection may be species-specific rather than being conserved.

The C-terminal core of TFIIB retains as much capacity to enter a pre-formed

TATA-TBP or TATA-TBP-TFIIA complex as the full-length protein, but blocks further

PIC assembly in vitro (Malik et al., 1993). Consequently, the core domain is

functionally defective in both basal and activated transcription (Malik et al., 1993), and

squelches the function of wild-type TFIIB probably due to the competition for binding

to TBP. Although Pol II is able to interact with the C-terminal end of the second repeat

of the core as has been shown in a pull-down assay (Ha et al., 1993), this interaction

apparently is not sufficient to recruit the enzyme into the PIC in vitro.

Given the essential function and high degree of conservation of TFIIB proteins,

one would expect that they might cross-function in different organisms. Indeed, the

arabidopsis TFIIB is able to replace hTFIIB to support AdML-dependent transcription

by HeLa nuclear extract in vitro (Baldwin and Gurley, 1996). A comparison of dTFIIB

and hTFIIB in a Drosophila embryo-derived in vitro transcription system showed the

same function by the two proteins on three core promoters: Adh, Jockey and AdE4

(Wampler and Kadonaga, 1992). These results suggest that TFIIBs are functionally

exchangeable among higher eukaryotic organisms. However, studies in yeast cells

showed contradictory results, in which both hTFIIB and arabidopsis TFIIB failed to

substitute for yeast TFIIB to support cell growth (Baldwin, 1997; Shaw et al., 1996).








Further analysis of the function of human-yeast hybrid TFIIB proteins has revealed a

specie-specific domain for yTFIIB (Shaw et al., 1996). This domain is part of the B1

helix of the first repeat. In addition to possible species specificity, TFIIBs also have

shown promoter-specific responses (Wampler and Kadonaga, 1992). For instance, with

the Drosophila Kr promoter, hTFIIB is unable to substitute in vitro for dTFIB.

Furthermore, dTFIIB enhances Kr, but suppresses AdE4 promoter activity in a dose-

dependent manner in the same transcription reaction. In contrast, the concentration of

dTFIIB has little effect on activated transcription driven by both VP16 and Spl

(Wampler and Kadonaga, 1992). The underlying mechanistic differences responsible for

these promoter-specific activities are still unknown.

As shown by many electrophoretic EMSA experiments, TFIIB is able to join the

TBP-DNA complex, but alone does not appear to have affinity for the DNA of the core

promoter. However, a recent study challenges this conclusion by showing that hTFIIB

has specific DNA binding activity (Lagrange et al., 1998). A binding-site selection

assay, which was previously applied in defining the consensus TATA sequence for TBP

binding (Wong and Bateman, 1994), was used to screen for any possible binding

preferences of TFIIB out of 1.7x107 DNA fragments (Lagrange et al., 1998). After two

rounds of selection, the 5'-G/C-G/C-G/A-C-G-C-C-3' sequence appeared to specifically

associate with TFIIB in terms of statistically significant frequency, and is defined as the

IIB recognition element (BRE) (Lagrange et al., 1998). The BRE is located at -32 to -38

base pair upstream of the TATA motif of the adenovirus major late promoter. The

guanine at -34 (5'-G/C-G/C-G/A-C-G-C-C-3') is thought to be the most critical base of

the BRE (Lagrange et al., 1998). The specificity ofTFIIB-BRE binding was confirmed








by substitution mutations in both DNA and protein. The D2 and E2 helices of the second

repeat of the core domain are believed to form the HTH-like motif for recognizing the

DNA major groove of the BRE. A double point-mutation in this motif, V283A/R286A,

inhibits both TFIIB/BRE binding and basal transcription (Lagrange et al., 1998).

Promoters with sequences less similar to the consensus BRE require eight times higher

TFIIB concentration than those with a consensus BRE to support the same level of basal

transcription (Lagrange et al., 1998). The protein-DNA cross-linking experiments

further showed that TFIIB interacts with the same BRE sequence in both the TATA-

TBP-TFIIB and TATA-TFIIB complexes (Lagrange et al., 1998). However, interactions

with the region downstream of the TATA are absent in the binary complex (TATA-

TFIIB) due to a lack of 800 DNA bending downstream of the TATA, which is a normal

distortion of DNA in the ternary complex (Kim and Burley, 1994). The discovery of the

BRE motif as a bonafide element of the core promoter suggests an important role for

TFIIB in promoter activity. Therefore, the recruitment of TFIIB may be a critical step in

transcription by promoters with no or weak BRE elements.

TBP-TFIIB Interaction


The TBP-TFIIB interaction is thought to be a critical step in the process of PIC

assembly. The EMSA experiments have indicated that the interaction is through the

conserved core domains (C-terminal) in both proteins (Hisatake et al., 1993). Detailed

amino acid contacts were further revealed by crystallography for the TATA-TBP2-

TFIIBc ternary complex (Nikolov et al., 1995). Although many amino acids from both

proteins are involved in numerous electrostatic, hydrogen bond and van der Waals

contacts, the primary interaction site is between the El helix of TFIIB and the C-








terminal stirrup of TBP. The El is a positive amphipathic helix, and the C-terminal

stirrup is negative in charge. It is likely that charge-charge interactions between the two

motifs account for the major binding energy. Amino acid substitution for either one of

the two glutamic acid residues at this stirrup totally abolished the formation of the

TATA-TBP-TFIIB complex in EMSA, demonstrating the critical roles of the two

negative residues in mediating the TBP-TFIIB interaction in both human and yeast (Lee

and Struhl 1997; Tang et al., 1996). However, since most of the studies on the TBP-

TFIIB interaction employed TBP rebound to DNA, it is not clear whether TBP with

DNA is the only conformation able to recognize TFIIB, or if the TBP-TFIIB interaction

can occur in the absence of DNA. Only one study used a pull-down assay to investigate

the direct interaction between hTBP and hTFIIB in the absence of DNA (Ha et al.,

1993). The results are in good agreement with the structural and EMSA data in that both

proteins used their C-terminal core domains to interact each other. Therefore, TFIIB is

able to recognize TBP with or without DNA.

The functional importance of the TBP-TFIIB interaction in transcription has

recently been tested by yeast genetic and the HeLa in vivo transcription system using a

sequential altered TATA-TBP-TFIIB specificity array. In yeast cells, the replacement of

the wild-type yTBP gene by its counterpart mutated in the C-terminal stirrup has little

effect on transcription by Gal4 and Gcn4 (Lee and Struhl, 1997). These TBP mutants do

not affect transcription driven by the pure recruitment of TFIIB using the fusion protein

LexA-TFIIB. Furthermore, in a system exhibiting pure recruitment of TBP, that is a

LexA-TBP fusion protein, these TBP mutants are functionally identical to the wild-type

protein in activating transcription from promoters containing the LexA binding site (Lee






25

and Struhl, 1997). Correspondingly, the TBP interaction mutants of TFIIB show no

impairment in supporting transcription by several activators (Chou and Struhl, 1997).

Interestingly, one yTFIIB point-mutation, R64E, significantly enhances formation of the

TATA-TBP-TFIIB complex, yet greatly inhibits both the basal and activated

transcription (Bangur et al., 1997). Although some of these mutants affect yeast cell

viability somewhat, all of these results strongly suggest that the TBP-TFIIB interaction

is not generally critical for transcriptional activation in yeast cells. Therefore, the

association of holoenzyme and TFIID in yeast cells may be mediated in large part by

other types of interactions between the two complexes outside of the TBP-TFIIB

interaction.

Unlike in yeast, in human cells the TBP-TFIIB interaction appears to be vitally

important for activated transcription. The same C-terminal stirrup mutations ofhTBP

abolished basal (Bryant et al., 1996) and activated transcription in both HeLa and COS

cells (Bryant et al., 1996; Tansey and Herr, 1997). In addition, the inhibitory action of

these TBP mutants on transcription activated by VP16 and CTF activation domains is

suppressed by the reciprocal mutation, R169E, of hTFIIB (Tansey and Herr, 1997). In

this case, the charge-charge interaction between TBP and TFIIB was presumably

restored by the R169E mutation of hTFIIB. In contrast to the findings from yeast

systems, these results suggest that in human cells, the TBP-TFIIB interaction performs a

critical role in supporting PIC assembly and activated transcription. However,

transcription driven by Spl is not significantly affected by the same TBP mutations

(Tansey and Herr, 1997). It seems that the requirement for the TBP-TFIIB interaction is

not universal, since recruitment mechanism of the PIC components by Spl somehow








apparently is able to bypass the requirement of the TBP-TFIIB interaction. As the final

comparison, VP16 activated transcription is not affected in yeast expressing TBP or

TFIIB mutants that impair the TBP-TFIIB interaction, although these same mutations

totally block VP16-activated transcription in HeLa cells (Chou and Struhl, 1997; Tansey

and Herr, 1997). Clearly, different organisms have differential preference for which

interactions (TBP-TFIIB, TBP-CTD, TFIIB-TAF, and so on.) to be used in the PIC

assembly and transcriptional activation. In addition, this preference can be activator-

dependent, as shown by Spl.

Interactions of TBP and TFIIB with Transcriptional Regulatory Proteins


Interaction with Activators

General transcription factors TBP and TFIIB have been reported to be the targets

of PIC recruitment for many activator proteins with different types of activation

domains, including acidic, glutamine-rich and proline-rich domains. The acidic

activators VP16 and GAL4 interact with both TBP and TFIIB (Kim et al., 1994a;

Roberts et al., 1993; Wu et al., 1996). Deletion analysis for hTFIIB protein has shown

that VP16 interacts in vitro with TFIIB at the C-terminal ends of the both core repeats,

involving the two helices El and E2, respectively (Roberts et al., 1993). Amino acid

substitution mutations within the El helix greatly inhibit the binding to VP16 (Roberts

et al., 1993). The bulky hydrophobic residue F442 in the VP16 activation domain was

shown to be critical for both TFIIB binding (Lin et al., 1991) and transcriptional activity

(Cress, and Triezenberg, 1991), providing an excellent correlation between the potential

for TFIIB recruitment and transcriptional activation. The interactions of TBP with the

acidic activation domains are also highly specific, as demonstrated by the loss of ability






27

ofyTBPLI 14K mutant to interact with VP16 and GAL4 activation domains (Kim et al.,

1994a; Melcher and Johnston, 1995). In addition, the level of GAL4-activated

transcription in vivo is positively correlated with the affinity in vitro for TBP and TFIIB,

suggesting that the recruitment of TBP and/or TFIIB is critical in the activated

transcription in yeast (Wu et al., 1996). The CTFI proline-rich activation domain

interacts with TFIIB, but not TBP, to facilitate the entry of TFIIB into the TATA-TBP

complex (Kim and Roeder, 1994). In addition, the glutamine-rich activation domain of

ftzQ interacts with TFIIB in vitro at its N-terminal zinc-ribbon domain. The biological

significance oftheftzQ-TFIIB interaction was implied by experiments where deletion of

the C-terminal core of TFIIB severely squelchedftzQ activity in transcription in vivo

(Colgin et al., 1995).

The interaction between an activator protein and TBP or TFIIB not only provides

a mechanism for recruitment of the latter to the core promoter, but also facilitates, in a

reciprocal manner, binding of the activator to its upstream element. These cooperative

effects between the activator binding to an enhancer sequence and PIC assembly on the

core promoter have been observed in several cases, including the hsp70 promoter

(Mason and Lis, 1997), GAL4 (Vashee and Kodadek, 1995), p53 (Chen et al., 1993) and

MyoD (Heller and Bengal, 1998) activators. These studies showed that the bindings of

the activators to the DNA elements were all enhanced by the interactions of the

activators with TBP. Additionally, the interaction between an activator and TBP or

TFIIB can alter the conformations for both interacting proteins. Proteolytic analysis of

the TFIIB protein showed significantly different patterns ofprotease digestion before

and after binding to VP16 (Roberts and Green, 1994). In vitro, TFIIB itself is a compact








molecule with tight folding between the N- and C-terminal domains. The binding of

VP16 to TFIIB disrupts the intramolecular interaction to expose the buried surfaces of

TFIIB so that it has more surfaces available for other protein-protein interactions. At the

same time, VP16 experiences a transition from a loose to an ordered structure when it is

completed with TBP or TFIIB (Shen et al., 1996). The induced structure of VP16 may

further stabilize its interactions with target proteins within the PIC. It is unclear how

important conformational alterations both in the target proteins and in the activation

domains seen in vitro are in living cells; however, the possibility that conformational

alterations play a major role must always be considered in discussion of activated

transcription.

To further investigate the roles of recruitment of TBP and TFIIB in PIC

assembly and transcriptional activation, many studies have been carried out in yeast and

human cells with an artificial recruitment approach (Chatterjee and Struhl, 1995;

Gonzalez-Couto et al., 1997; Klages and Strubin, 1995; Lee and Struhl, 1997; Xiao et

al., 1997; Xiao et al., 1995b). Instead of being recruited by an activation domain, TBP

or TFIIB is translationally fused directly to a DNA binding domain and forced to

localize to the promoter when the attached DNA binding domain is bound to its

recognition site. This system provides for pure recruitment of only one factor, either

TBP or TFIIB, to directly address whether or not this simple recruitment can lead to PIC

assembly evidenced by transcriptional activation. The results obtained from these

studies have demonstrated that the simple recruitment of either TBP or TFIIB alone is

sufficient to facilitate PIC assembly on the promoter resulting in the activation of

transcription. Activation of transcription by fusing a DNA binding domain to TBP or






29

TFIIB strongly support models for activated transcription that incorporate recruitment of

general transcription factors through protein-protein interactions between activator

proteins bound to the promoter and their target proteins within the PIC, and specifically

support conclusions that TBP and TFIIB can serve as crucial targets for activation

domains.

Despite good evidence for the recruitment model for activated transcription, it is

important to note that a potential for interaction demonstrated in vitro between an

activator protein, or motif, and a general transcription factor does not necessarily imply

that this potential for interaction will actually be important, or crucial in activating

transcription in a living cell. For example, mutations of either TBP or TFIIB defective of

interacting with VP16 had no effect on transcription activated by VP16 in vivo (Chou

and Struhl, 1997; Tansey and Herr, 1995). These results suggest that transcriptional

activation by VP16 may be mediated by recruitment pathway(s) not involving either

TBP or TFIIB. This apparently paradoxical finding may be due to the ability of VP16 to

bind to multiple PIC members like TAFs (Goodrich et al., 1993), TFIIH (Xiao et al.,

1994), TFIIA (Kobayashi et al., 1995) and co-activator PC4 (Ge and Roeder, 1994) in

addition to TBP and TFIIB, as shown by in vitro biochemical studies. Given such

possible multiple interactions in vivo, one would not expect a dramatic affect on

transcription by the disruption of a single interaction because of the redundant

recruitment pathways. Theoretically, TFIID and holoenzyme can be readily recruited by

an interaction with any one of the components.

In some cases, a direct interaction between TBP or TFIIB and a particular

activation domain is not sufficient for transcriptional activation. Activator proteins in






30

this category are best described as weak activators. They are believed to be able to

support the initiation, but not the elongation of transcription (Blau et al., 1996). The

basis for this differential effect on initiation versus elongation is not understood at

present; however, the ability for an activation domain to interact with and, therefore, to

recruit TFIIH seems to be important for its activity in supporting the elongation of

transcription (Blau et al., 1996; Kumar et al., 1998).

Transcriptional activation by a particular activator may be enhanced or

suppressed by elevated levels of TBP or TFIIB proteins. When TBP or TFIIB is limiting

in a transcription system, the over-expression of TBP or TFIIB will enhance its

interaction with the activator and stimulate transcription. In contrast to this positive

effect, TBP and TFIIB also display a negative effect resulting from the squelching of

productive interactions with the activator. In this case, excess amounts of TBP and/or

TFIIB are unable to incorporate into the PIC and form nonfunctional complexes with the

activation domain to inhibit transcription. However, these effects often appear to be

activator-dependent. In the same transcription system in HeLa cells with multiple assays

for different activation domains, over-expression of TBP stimulated transactivation by

VP16, Tat, or estrogen receptor (ER), but inhibited transcription activated by Spl or NF-

1 (Sadovsky et al., 1995). The underlying mechanisms for these different responses by

activators are still unknown, but could be due to their differences in TBP affinity.

Sometimes, these positive or negative effects can also be cell-type-dependent as shown

by the vitamin D receptor (VDR), with which over-expression of TFIIB stimulated

VDR-dependent transcription in P19 embryonal carcinoma cells, but suppressed VDR

activity in NIH 3T3 cells, both in a dose-dependent manner (Blanco et al., 1995). It is








not known whether the difference seen between the two cell line is due to participation

of different co-factors in transcription as suggested (Blanco et al., 1995).

Interactions with Repressors

Compared to activator proteins, functional mechanisms for repressor proteins are

much less clear. In general, however, interactions with PIC componentss, like TBP

and/or TFIB, may also be crucial to the mechanism of transcriptional repression. As

opposed to activators, repressors can prevent PIC assembly by interacting with TBP

and/or TFIIB. For instance, the E1A repression domain has been shown to bind to TBP

and disrupt TBP-TATA interaction. This effect can be reversed by TFIIB in vitro (Song

et al., 1997). The Dorsal Switch Protein (DSP1) inhibits TFIIA-TBP complex formation

by interacting with TBP (Kirov et al., 1996). The C-terminal repression domain of Eve

protein simply inhibits TFIID's ability to recognize the TATA element by binding to

TFIID (Austin and Biggin, 1995). The Negative co-factor 2 (NC2) prevents TFIIB from

joining with the TBP-promoter complex by interacting directly with the promoter DNA

(Goppelt et al., 1996). The interaction of NC2 to DNA probably blocks the DNA surface

for the recognition by TFIIB. It appears that different repressors are able to prevent PIC

formation at different stages of the assembly; some blocking further assembly of the PIC

by masking protein-protein interactions, and others by interfering with DNA-protein

interactions. In addition, like co-activators, co-repressors may be required for some

repressors to function (Mermelstein et al., 1996). Overall, the details of interaction

within the PIC that distinguish proteins that activate versus those that repress

transcription are still poorly understood.








Transacting Proteins


A typical transcriptional activator protein contains two functional domains: the

DNA binding domain and the transcriptional activation domain. The two domains often

are separable from each other and can usually reconstitute transcriptional activity when

any of the two types are fused together. In my study using plant cells, the DNA binding

domain from the GAL4 activator protein (Marmorstein et al., 1992) was translationally

fused to the activation domains from several other sources to activate transcription of a

reporter gene containing GAL4 binding sites upstream of the TATA motif The chimeric

activators used in this study include the well characterized activation domains ofVP16

from human herpes simplex virus, GAL4 from yeast and Ftz from Drosophila as well as

several others from plants with less characterized features including VP1 from maize,

HSF from tomato and a 14-3-3 protein from arabidopsis. The following sections attempt

to summarize the important features for each activator protein (or potential activator

protein) used in this study.

GAL4

The GAL4 protein is the transcriptional activator required for expression of the

galactose/melibiose regulon in yeast in order to regulate carbohydrate metabolism

(Laughon and Gesteland, 1982). The protein consists of 881 amino acids. The N-

terminal 1-65 aa comprises the DNA binding domain (DBD) recognizing the GAL4 site

in the upstream activation sequences of the structural genes of the regulon (Marmorstein

et al., 1992). The C-terminal portion of the protein contains two activation domains

(AD) (Ma and Ptashne, 1987b), and the large middle portion of the protein can be

deleted without affecting transcription (Ma and Ptashne, 1987b). The transcriptional






33

function of the GAL4 protein is down-regulated by the GAL80 protein in uninduced

cells induciblee by galactose), which binds to the 30 aa at the extreme C-terminal region

of the GAL4 AD (Ma and Ptashne, 1987a). This inhibition can be reversed by

phosphorylation of the GAL4 protein (Parthun and Jaehning, 1992).

The GAL4 DBD consists of two functional motifs: the cysteine-rich region (aa

10-35) and two dimerization domains (aa 50-65 and aa 65-94). In the DBD dimer, each

of the two "zinc-finger"-like domains interacts with the major groove of the consensus

GAL4 site (5'-CGGAGGACTGTCCTCCG-3') at the CGG triplet of either end

(Marmorstein et al., 1992). A peptide containing the DBD and a single dimerization

domain (aa 50-65) is able to bind DNA, but this interaction is further enhanced by

inclusion of the second dimerization domain (aa 65-94). Because of the high binding

specificity between DNA and the GAL4 DBD, and the lack of recognition of the GAL4

binding site outside of yeast, the GAL4 transcription system has been widely used in

many heterologous organisms to characterize transcriptional activity of many activation

domains. This approach has also been very successful in plant transient expression

systems in characterizing the potential activation domains of plant activator proteins

(Bobb et al., 1995; Nakayama et al., 1997; Schwechheimer et al., 1998; Ulmasov et al.,

1995; Urao et al., 1996).

The 41 aa AD at the C-terminal end of the GAL4 protein is rich of acidic

residues and is classified as an acidic activation domain. Unlike the VP16 AD, this

domain is thought to form a P-sheet structure instead of the amphipathic a-helix

(Leuther et al., 1993). However, the mutagenesis analysis on the GAL4 AD showed that

the acidic amino acids present in the motif were not important for activity (Leuther et






34

al., 1993). The AD still retains full transcriptional activity when the acidic amino acids

are replaced by a cluster of positive residues (Leuther et al., 1993). In yeast cells, over-

expression of GAL4 protein squelched transcription of the genes without the GAL4 site,

a phenomenon believed to be due to the titration of the general transcription factors)

(Gill and Ptashne, 1988). Indeed, the GAL4 AD is able to interact with both TBP and

TFIIB, and transcription likely is mediated by these interactions in vivo (Wu et al.,

1996).

VP16

VP16 is a human herpes simplex virus (HSV) protein that is the transcriptional

activator for the five immediate early genes of the virus. The protein itself does not have

affinity for promoter DNA, instead, it relies on other transcriptional factors of the human

host cells (Triezenberg et al., 1988a). Promoter binding activity is provided by the host

protein Oct-1, which recognizes the 5'-TAATGARAT-3' sequence of the viral promoter

(Ster et al., 1989). However, the binding of Oct-1 to the promoter does not lead to

transcriptional activation (Triezenberg et al., 1988b). Transcription can not occur until

VP 16 joins to Oct-1 with the assistance of other proteins called host cellular factors

(HCF) to form the multiprotein-DNA complex (Lai and Herr, 1997). The

phosphorylation of S375 of VP16 outside the activation domain by CKII is required for

the assembly of the activator complex in vitro and further the transactivation by the

complex in vivo (O'Reilly et al., 1997).

The strong acidic activation domain of VP16 is located at the end of the C-

terminus of the protein from aa 413-490 (Cousens et al., 1989). The activation domain

can be further divided into two subdomains: one at aa 413-456 and the other from aa






35

457-490. Each of the two subdomains is able to independently activate transcription

(Lyons and Chambon, 1995). Mutational analysis of the activation domain indicates that

the bulky hydrophobic residues are more important than the acidic residues for

transcription (Regier et al., 1993). While some activation domains stimulate

transcription only at the initiation step, VP16 can activate transcription at both initiation

and elongation stages (Brown et al., 1998). The critical residue F442 appears to only be

important for initiation with the other residues involved in a postinitiation step. These

two activities of VP16 have been shown to function synergistically in transcription in

vivo (Ghosh et al., 1996). The strong transcriptional activation elicited by VP16 is

thought to be related to its ability to interact with and recruit multiple general

transcription factors including TBP (Kim et al., 1994a), TFIIB (Roberts et al., 1993),

TFIIA (Kobayashi et al., 1995), TFIIH (Xiao et al., 1994), TAF,,s (Goodrich et al.,

1993) and PC4 (Ge and Roeder, 1994). Consistently, it is not surprising that high levels

of VP16 protein expression result in transcriptional squelching, since numerous target

proteins may be sequestered in nonfunctional complexes (Berger et al., 1990; Natesan et

al., 1997).

Ftz

Ftz is a Drosophila transcriptional activator protein involved in segmentation

development during embryogenesis (Kuroiwa et al., 1984), and is comprised of 413

amino acid residues. The N-terminal half of the protein contains the homeodomain,

which is similar to the HTH DNA binding domain ofprokaryotic DNA-binding proteins

(Desplan et al., 1988). This domain recognizes the 5'-CC/AATTA-3' motif in the

promoters of the segmentation genes such as engrailed (en). The C-terminal half






36

contains the glutamine-rich activation domain ftzQ (aa 349-408), which also functions

as an activator when fused with the GAL4 DBD in yeast cells (Fitzpatrick and Ingles,

1989). In different protein-DNA complexes, ftz can either activate en (Fitzpatrick and

Ingles, 1989) or repress wg (Ingham et al., 1988) expression. The specific activity of ftz

can be regulated by its phosphorylation state (Dong et al., 1998; Krause and Gehring,

1989) and by its positive co-factor Ftz-F1 (Yu et al., 1997), which is a member of the

nuclear hormone-receptor superfamily. Transcriptional activation by ftzQ is believed to

be mediated by its interaction with TFIIB at the zinc-ribbon domain. The functionally

defective N-terminal region of TFIIB severely squelches the ftzQ activity (Colgan et al.,

1993), and the mutations, H17S and C33S, at the zinc-ribbon suppresses the squelching

and restores the ftzQ activity (Colgin et al., 1995).

VP1

VP1 is a maize transcriptional regulatory protein involved in seed maturation and

anthocyanin biosynthesis during seed development. It activates seed maturation-related

gene(s) expression such as Em, but represses seed germination-related genes such as a-

amylase genes (Hoecker et al., 1995). The protein contains a total of 691 residues with

the N-terminal 121 aa comprising the activation domain. This domain is rich in serine

and proline in addition to negatively charged amino acids and is predicted to form two

amphipathic a-helices similar to that of the VP16 activation domain (McCarty et al.,

1991). This region can be deleted without affecting the repression function of VP1

protein (Hoecker et al., 1995). The B3 domain located at the C-terminal half ofVP1 is

the DNA binding domain for the Sph element in the Cl promoter (Suzuki et al., 1997).

VPI also activates transcription by the G-box element, presumably through protein-






37

protein interactions with other DNA binding factors such as EmBP1 (Hill et al., 1996).

The targets for the VP1 activation domain in the PIC are unknown. However, in vitro

binding experiments indicate that it interacts with TFIIB (Baldwin, 1997).

LpHSF8

LpHSF8 is one of the three heat shock transcription factors known in tomato

(Scharfet al., 1990). It is believed that the HSF proteins pre-exist in the cytosol, and are

localized to the nucleus upon heat stress (Scharf et al., 1998). DNA binding activity is

located in the N-terminal portion of the proteins, and a domain responsible for

trimerization (trimerization domain) is adjacent to the DBD at its C-terminal end. HSF

proteins are able to bind to the heat shock-response element of the heat shock genes,

such as hsp70, only in the trimerized state (Kroeger et al., 1993). The tomato HSF8 also

contains an activation domain ofaa 394-527 at its C-terminal region rich in acidic

residues interspersed by bulky hydrophobic residues (Treuter et al., 1993). Although

Drosophila HSF has been shown to interact with TBP in vitro (Mason and Lis, 1997),

no information regarding targets with respect to plant HSFs has been reported.

The 14-3-3 Proteins

The 14-3-3s are highly conserved eukaryotic proteins with multiple functions. In

addition to their participation in different signal transduction processes (Braselmann and

McCormick, 1995; Dellambra et al., 1995; Freed et al., 1994; Gelperin et al., 1995),

circumstantial evidence suggest that 14-3-3 proteins may also be involved in

transcriptional regulation in both plant and human systems. Although the 14-3-3

proteins alone do not bind to DNA, several studies have demonstrated that at least one

plant 14-3-3 protein is associated with the G-box DNA binding activity of nuclear








extracts from arabidopsis (Lu et al., 1992), maize (de Vetten and Ferl, 1994) and rice

(Schultz et al., 1998). Consistent with a role in regulating gene expression, 14-3-3

isoforms are found in the nuclei of both arabidopsis and maize cells in vivo (Bihn et al.,

1997). More recently, different 14-3-3 isoforms were found to directly interact with two

G-box-related transcription factors, VP1 and EmBPI, with different affinities (Schultz et

al., 1998). In animal systems, human 14-3-3 ir is found to interact with another

transcription factor, glucocorticoid receptor, and stimulate glucocorticoid receptor-

dependent transcription in COS-7 cells (Wakui et al., 1997). It is possible that 14-3-3

proteins may affect transcription as part of a DNA-protein complex that may associate

with other transcriptional regulatory proteins to either regulate the activity of the

complex, or directly contact the PIC components and influence the PIC assembly.

Rationale for the Present Study


Transcriptional regulation has been an extensive subject of both human and yeast

research for more than a decade. However in plants, related research has mostly limited

to the characterization of promoter elements. There has been little information available

to help design a high-output-expression system or provide specific control of gene

expression at the level of manipulating the function and activity of the transcription

machinery. The results from studies in human and yeast systems sometimes appear to be

controversial, suggesting that different kingdoms may have unique aspects of

transcriptional regulation in addition to the common requirements. These considerations

resulted in a strong encouragement to initiate this project to study the functional roles of

plant TBP and TFIIB proteins, which appear to be the most effective factors for control

of PIC function. The following chapters will describe the findings of this study.














CHAPTER 2
CRITICAL ROLE OF THE TBP-TFIIB INTERACTION IN
SUPPORTING ACTIVATED TRANSCRIPTION IN PLANT CELLS

Introduction


Transcription of class II genes in eukaryotic cells is directed by the interaction

between two megacomplexes of proteins, TFIID and RNA polymerase II holoenzyme.

TFIID provides promoter-binding activity, and the holoenzyme contains the catalytic

function for mRNA synthesis. The interaction of these two complexes results in

formation of the PIC, the final configuration of factors before the initiation of

transcription. Therefore, mechanisms that facilitate the recruitment of TFIID and

holoenzyme to the promoter enhance formation of the PIC and lead to gene activation.

Although activator proteins usually function by binding to the promoter and then to

general transcription factors to recruit them to the promoter, general transcription factors

can also be directly tethered to the promoter by translational fusions with heterologous

DNA binding domains (DBDs). In yeast cells, the pure recruitment of either TFIID or

holoenzyme to the promoter by directly tethering TAF,,s, or TFIIB can lead to high

levels of transcription comparable to that achieved by a strong activator such as VP16

(Gonzalez-Couto et al., 1997). In this tethered system, recruitment of the first complex

to the promoter presumably results in the subsequent recruitment of the second complex.

Apparently, recruitment of the second complex is mediated by interactions between

components of TFIID and the holoenzyme. Biochemical studies in vitro have indicated








that the association of TFIID and holoenzyme on the promoter involves many protein-

protein contacts including those between TBP and TFIIB (Buratowski and Zhou, 1993;

Nikolov et al., 1995), TBP and TFIIA (Geiger et al., 1996; Tan et al., 1996), TBP and

the CTD (Usheva et al., 1992), dTAFO4 and TFIIB (Goodrich et al., 1993), hTAF80

and TFIIEa (Hisatake et al., 1995), hTAF180 and RAP74 (TFIIF) (Hisatake et al.,

1995), and hTAF1250 and RAP74 (Ruppert and Tjian, 1995). However, since these

studies were based on interaction assays performed in vitro using isolated components,

the relative importance and strength of each individual interaction in the context of the

PIC is not known. It is unclear whether each of the potential interaction pathways

between TFIID and holoenzyme is critical for activated transcription, or if their relative

importance is context sensitive.

In recent years, the TBP-TFIIB interaction has been studied intensively,

primarily because these two factors are crucial to both basal and activated transcription.

EMSA experiments have shown that the N-terminal region of TFIIB is dispensable for

TBP binding without affecting the affinity of interaction (Hisatake et al., 1993). For this

reason and its lack of defined crystal structure, only the C-terminal core domain of

hTFIIB (aa 112-316; hTFIIBc) was used in the X-ray crystallographic analysis of

protein structure for the TATA-AtTBP2-hTFIIBc ternary complex (Nikolov et al.,

1995). This analysis revealed that TFIIBc recognizes TBP from beneath the TBP-DNA

complex and, together with TBP, forms a partial clamp around the TATA motif (Fig. 2-

1). In addition, TFIIB contacts both ends of the TATAA to further stabilize the complex.

The overall interactions between TBP and TFIIB involve eight residues from TBP

(E131, Y135, Y143, E144, P145, E146, L147 and K197) and twelve residues from










































Figure 2-1. TBP-TFIIB-DNA ternary complex (Nikolov et al., Nature 377:119-
128, 1995), showing location of amino acids residues involved in close contacts
between TBP and TFIIB. Mutations were placed in arabidopsis TBP2 at E144R,
E146R and K197E in this study.






42

TFIIB (Y165, R169, T176, F177, K188, G192, F195, N207, L208, D243, S249 and

P250). Four out of eight residues from TBP are located in the C-terminal stirrup and

include the following: E144, P145, E146 and L147. These four stirrup residues make

contacts with ten amino acids ofhTFIIBc to form two salt bridges, two H-bonds, and

seven van der Waals interactions in the ternary complex. Residue E146 makes the most

contacts with TFIIB by forming the strongest salt bridge, two H-bonds and one van der

Waals interaction. It seems likely from this structural information that aa E146 of

arabidopsis TBP2 is the most critical residue for TFIIB binding.

Protein sequence comparisons suggest that the TBP-TFIIB interaction is highly

conserved among eukaryotic organisms. The sequence for the C-terminal stirrup of TBP

is identical among arabidopsis (Kim and Burley, 1994), human (Kao et al., 1990),

Drosophila (Muhich et al., 1990) and yeast (Horikoshi et al., 1989) proteins. Out of the

twelve TBP binding residues ofhTFIIBc, all are conserved in dTFIIB, ten in AtTFIIB,

and eight in yTFIIB (Baldwin and Gurley, 1996). The importance of the C-terminal

stirrup of TBP in TFIIB interaction has been confirmed by mutational analysis ofhTBP

and yTBP. In hTBP, substitution ofE284, E286, or L287 (corresponding to E144, E146

and L147, respectively, in AtTBP2) by alanine reduces the affinity for TFIIB to about

5% of the wild type level in vitro (Tang et al., 1996). The same alanine substitutions of

yTBP result in 100-, 50-, and 10-fold reductions in TFIIB binding, respectively (Lee and

Struhl, 1997). These stirrup mutations specifically disrupt the interaction of TBP with

TFIIB, but do not affect those with TFIIA, TFIIF, Pol II, TFIIE and TFIIH (Tang et al.,

1996). Although similar studies have not been done in Drosophila or in plants, one






43

would expect that the C-terminal stirrup of TBP also serves as a critical TFIIB binding

domain during the PIC assembly in these systems as well.

The "altered specificity" system is a powerful approach used in defining

functional roles of the TBP-TFIIB interaction in human systems. This system is

established by the mutations in both the TATA-box and TBP. This specificity was first

identified in yeast by using the TGTA-his3 gene to select for a TBP mutant that was

able to support cell growth (Strubin and Struhl, 1992). The mutant TBP, TBPm3, has

three substitutions for the residues located in its concave DNA binding surface as

follows: I194F, V203T and L205V. TBPm3 possess an extended DNA binding

specificity from TATAA to TGTAA, to which the wild type TBP is unable to bind as

demonstrated by EMSA experiments (Strubin and Struhl, 1992). Therefore, transcription

of a reporter gene containing the TGTA instead of the TATA motif would be dependent

on TBPm3, but not the endogenous TBP. This system allows precise measurement of

the transcriptional effect of additional mutations on TBPm3 without interference from

the endogenous TBP protein in a transient expression assay. The altered specificity of

TBPm3 has also been confirmed in studies with hTBP (Bryant et al., 1996) and AtTBPs

(Heard et al., 1993).

The effects of the C-terminal stirrup mutations of TBP on transcription were

assessed using this TBPm3/TGTA coupled system. The three single substitution

mutations, E284R, E286R and L287E, of the C-terminal stirrup of hTBP abolished basal

transcription in vitro (Bryant et al., 1996). In addition, E284R and L287E mutations

greatly inhibited activated transcription by VP16 or EIA in COS cells, while the E286R

mutation showed a moderate reduction (about 50%) in transcription (Bryant et al.,






44

1996). Similar studies done in HeLa cells showed that E284R and E286R totally

abolished activated transcription by CTF or VP16, but mutation L287A resulted in much

less inhibition (Tansey and Herr, 1997). Using VP16 activity for comparison, the

importance of each individual residue in the C-terminal stirrup ofhTBP in activated

transcription appears to be cell-type-dependent, although all are required for in vitro

binding in EMSA assays. To further support the conclusion that transcriptional

impairment caused by stirrup mutations is due to the disruption of the TBP-TFIIB

interaction, the reciprocal mutation in hTFIIB, R169E, was shown to specifically

suppress the inhibitory effects of the E284R mutation, presumably by restoring the

charge-charge interaction between TBP and TFIIB (Tansey and Herr, 1997).

Not only are there differences in the relative importance of individual amino

acids (HeLa vs. COS cells), dependence on the TBP-TFIIB interaction for activated

transcription may also vary between different activators. For example, the Spl activator

is able to tolerant C-terminal stirrup mutations with showing only 50% reduction in

transcription when assayed in HeLa cells (Tansey and Herr, 1997), whereas VP16, CTF

and p53 are highly sensitive to the same mutations. Differences in dependence on the

TBP-TFIIB interaction suggest that some activators may use different interaction

pathways for PIC assembly.

Surprisingly, the results obtained by yeast genetic approaches are contrary to

those obtained in human transient expression studies. When yTBP was fused to the

LexA DBD, high levels of transcription were achieved in the absence of an activator

protein due to the direct tethering of TBP to the promoter. This system, which

circumvents the need for activators, was used to evaluate mutations in the C-terminal






45

stirrup of yTBP in order to eliminate potential stabilizing effects between an activator

and components of TFIID or the holoenzyme. Stirrup mutations E186A, E188A and

L189A in LexA-yTBP were all able to support high levels of transcription indicating

that a functional yeast PIC can be assembled without involvement of an activation

domain, even when the TBP-TFIIB interaction is disrupted by mutation (Lee and Struhl,

1997). Moreover, both E188A and L 189A mutations supported transcription dependent

on LexA-Gcn4 and LexA-yTFIIB as efficiently as the wild type yTBP (Lee and Struhl,

1997). In a separate study, yTFIIB mutants were created that were incapable of binding

to yTBP and VP16 (Chou and Struhl, 1997). These TFIIB mutants supported

transcription by a series of different activation domains with both synthetic and natural

promoters as efficiently as wild type TFIIB. Paradoxically, a yTFIIB mutation

enhancing TATA-TBP-TFIIB ternary complex formation failed to support basal and

activated transcription in vitro; however, this mutation did support activated

transcription in vivo at lower levels (Bangur et al., 1997). These results strongly argue

that the TBP-TFIIB interaction may be either redundant to other interaction pathways, or

nonessential, in the process of PIC assembly in yeast cells. Overall, it is apparent that

the TBP-TFIIB interaction in yeast is not as critical as in humans with respect to

supporting PIC assembly and transcription.

In plants, although both TBP and TFIIB cDNAs have been isolated, few studies

have examined function or addressed questions related to the roles of protein-protein

interactions in the process of activated transcription. In this chapter, the functional

importance of the TBP-TFIIB interaction is characterized in both basal and activated

transcription by transient expression assays in maize cells. Maize suspension cells and






46

protoplast transient expression systems have been successfully used to study the activity

of a growing number of transcription factor proteins in plants (Goffet al., 1992; Marrs

et al., 1993; McCarty et al., 1991; Yanagisawa and Sheen, 1998). In this study, TFIIB

binding mutants of AtTBP2 were over-expressed in maize suspension cells, and the

effects of these mutants on transcription were determined by monitoring reporter gene

activities.

Materials and Methods


Mutagenesis for AtTBP2

The Altered Site II mutagenesis system of Promega was used to generate amino

acid substitution mutations for AtTBP2 in the Ex-1 vector by the manufacturer's

protocol. The targeted residues were E144, E146 and K197. These three amino acids are

predicted to be involved in three charge-charge interactions between TBP and TFIIB in

the TATA-TBP-TFIIBc ternary complex based on the structure determination of the

AtTBP2/hTFIIB complex (Nikolov et al., 1995). Residues E144 and E146 are located in

the C-terminal stirrup, and K197 is located at the C-terminal end of AtTBP2. Each of the

three residues was substituted by an amino acid with the ionic charge opposite to its

wild-type counterpart to form single, double, or triple point-mutations as follows:

E144R, E146R, K197E, E144R/E146R and E144R/E146R/K197E. After confirmation

by DNA sequencing (Microbiology Department Sequencing Core, University of

Florida), both wild-type and mutant TBP cDNAs were released from the Ex-1 vector by

Sal I/BamH I digestion and subcloned into the pBI221 (Clontech) derived plant

expression vector using Sal I/Bgl II sites. The first two residues of TBP within the non-

conserved N-terminal loop were inverted as TM due to subclonings. Both AtTBP2 and






47

AtTFIIB recombinant proteins contained thirteen additional amino acid residues

(MASMTGGQQMGRS) at the N-terminal ends and two residues (EI) at the C-terminal

ends followed by a stop codon. The first eleven residues are the T7-epitope

(MASMTGGQQMG).

Particle Bombardment in Maize Suspension Cells

The reporter and effector plasmid DNAs were precipitated onto gold particles by

the following procedure: 35 gl of DNA was mixed with 37 gl of gold (40mg/ml) in a 1.5

ml Eppendorftube. 50 ll of 2.5 M CaCl2 and 20 .ll of 100 mM spermidine were

pipetted onto the side wall of the tube separately, then immediately mixed with the gold

particles by vortexing for 20 sec. After spinning in a mini-centrifuge (5 sec), the

supernatant solution was pipetted out and discarded. The DNA-coated gold particles

were washed in 200 pl of 100% ethanol by sonication for 5 sec using a mini-sonicator

bath, then collected by centrifugation for 5 sec, and re-suspended in a final volume of 80

ul of 100% ethanol. For DNA-gold preparations, the amount of individual plasmid

DNAs remained constant between experiments as follows: 2.5 pg for Ubi/LUC as

internal control, 2.5 lg for the GUS reporter driven by the CaMV 35S minimal promoter

with or without upstream elements, 2.5 ulg for Gal4 DBD fusion activators, and 10 lg

for T7, T7-TFIIB, T7-TBP (wt or mutants) effectors. The Ubi/LUC construct was driven

by a maize ubiquitin promoter previously described (Christensen and Quail, 1996). The

minimal promoter/GUS construct was under control of the CaMV 35S minimal

promoter (nt -46) (Odell et al., 1985), and used to measure the basal in vivo

transcription. The pBI221 vector was used to measure transcription activated by the wild

type CaMV 35S promoter in the 800 bp Hind III-BamH I fragment (Clontech). The








GAL4/GUS reporter was controlled by the synthetic GAL4 binding sites as described

(Verner and Gurley, in preparation). The effector constructs of either Gal4 DBD fusion

or T7-tag fusion were derived from the pBI221 vector as described (Verner and Gurley,

in preparation). Within each experiment, the total DNA was kept constant by including

an "empty" vector having only the T7-tag coding sequence driven by the CaMV 35S

promoter. Constructs for reporter and effector plasmids are illustrated in figures 2-2 and

2-3.

The DNA-gold particles were delivered into log-stage maize suspension cells

using a Bio-Rad particle bombardment apparatus with set at 1100 psi. For each

bombardment, 5 pl of the well-suspended DNA-gold particles was pipetted onto the

carrier disk and allowed to air-dry. Cell samples were prepared as follows: 50 ml of cell

culture was poured into a 50 ml sterile centrifuge tube and the cells allowed to settle by

gravity to a volume of 5 to 7 ml. The extra medium was discarded, resulting in a

cell:medium ratio of 1:1 (v/v). The cells were then well suspended, and 300 .l was

pipetted onto a 2.5 cm diameter circle Whatman filter paper previously placed on an MS

phytogel plate (Murashige and Skoog, 1962). After particle bombardment, the filter-

immobilized cells were allowed to recover for 20 to 22 hr in the dark at 260C. The cells

were harvested by grinding using a mortar and pestle for 1 min in 300 ul of extraction

buffer containing 200 mM Na2HPO4/NaH2PO4, pH 7.8, 4 mM disodium

ethylenediamine tetraacetate (EDTA), 2 mM 1,4-dithiothreitol (DTT), 5% glycerol and

1 mg/ml bovine serum albumin (BSA). The supernatants were collected by

centrifugation for 15 min at 12,000 rpm and aliquots assayed for GUS and luciferase

activities.














Ubi/LUC
(Internal control) -
Ubiquitin promoter


+1


Luciferase gene


Null
promoter/GUS




Minimal
promoter/GUS


No promoter




- TATAA
CaMV 35S (-46)


GUS gene


+1


GUS gene


+1


35S/GUS


- UAS


TATAA


CaMV 35S promoter


GAL4/GUS


TATAA
Gal4 (10x) CaMV 35S
(-46)


GUS gene


+1


GUS gene


Figure 2-2. Reporter plasmids: luciferase internal control and GUS test reporters.


















Gal4 DBD
fusions:


CaMV 35S promoter Gal4 AD:
DBD
ftzQ
Gal4
LpHSF8
VP1
VP16


T7-tag fusions: H


UAS


TATAA


CaMV 35S promoter


r+1




T7 effector:


wt AtTBP2
TBP mutants
wt AtTFIIB


Figure 2-3. Effector constructs: Gal4 DBD fusions and T7 epitope-tagged








GUS and Luciferase Assays

The luciferase assay was conducted using a multipurpose scintillation counter

(Beckman) to measure single photon emission from the enzymatic reaction in which 5

pl of maize cell extract was incubated with 25 ul of luciferase substrate (Promega) at

room temperature. Light emission was measured immediately after the mixing of extract

and substrate.

To measure GUS activity, 50 ul of the original extract was incubated with 75 rl

of 2 mM 4-Methylumbelliferyl P-D-Glucuronide (MUG) (GUS substrate) at 370C for

2.25 hours, and the reaction was stopped by mixing 50 pl of the extract/substrate

mixture with 950 pl of 0.2 N Na2CO3. The product of the enzymatic reaction 4-

Methylumbelliferone (MU) was measured using a spectrofluorophotometer (Shimadzu,

RF5000) with excitation wavelength at 365 nm and emission wavelength at 445 nm

respectively. The MU concentration for the first 15 min of the reaction was taken as the

background and subtracted from the final Mu concentration after the 2.25 hr reaction.

Protein Expression and Purification from E. coli

GST-AtTFIIB and GST-AtTBP2 or its mutants were expressed in E. coli using

the pGEX-2TK expression vector (Pharmacia). The overnight grown bacterial cells were

diluted 1:100 in 50 ml Luria Broth (LB) medium containing 100 pg/ml ampicilin, and

then allowed to grow for 3 hr at 370C. Protein expression was induced with 10 LM

isopropyl-1-thio-p-D-galactoside (IPTG) for 7 hr at room temperature. All IPTG-

induced E. coli cells were collected by centrifugation, washed with cold Ix PBS buffer,

and suspended in 1 ml of cold lx protein binding buffer. The lx protein binding buffer

contained 20 mM (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES),






52

pH 7.5, 0.1 M KCI, 5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 10% glycerol and 0.05%

NP-40.

For protein purification, the cells were disrupted by four one minute bursts of a

probe sonicator, then centrifuged for 30 min at 12,000 rpm. Soluble proteins in the

supernatants were purified with glutathione-agarose beads (Pharmacia) at 4C for 1 hr

with continuous rotation. The beads were pelleted by brief microcentrifugation, then

washed with cold lx protein binding buffer plus 0.5 M KCl for 10 min, followed by a

final wash with cold lx protein binding buffer, and then suspended in the same buffer.

The quantity of each individual GST fusion protein was determined by comparison to

BSA standard protein in Coomassie stained sodium dodecyl sulfate (SDS)-PAGE.

In vitro Protein Translation

The pGEM-3z vector (Promega) was engineered to have the T7-epitope coding

sequence directly fused with the start codon. Coding sequences of AtTFIIB and AtTBP2

or its mutants were cloned into the modified pGEM-3z vector to express the T7-tagged

proteins in vitro. T7-TFIIB was expressed by a transcription/translation coupled (TNT)

wheat germ system, and T7-TBP was expressed by the rabbit reticulocyte TNT

(Promega), using the manufacturer's protocol except that TBPs were expressed at room

temperature.

In vitro GST Pull-Down Assay

To examine the AtTBP2-AtTFIIB interactions, T7-TBP or mutants were

incubated with bead-immobilized GST-TFIIB (15 pg), or reciprocally, T7-TFIIB with

equal amount (15 pg) of GST-TBP or its mutants (the total amount of beads was kept

the same for each binding reaction by adding buffer washed blank glutathione beads








when necessary). Binding reactions were for 1 hr at 40C in 300 gl of lx protein binding

buffer containing 0.1% BSA in a continuously rotated tube. The beads were then

extensively washed with Ix protein binding buffer (4 times, 1 ml each). The bound

protein molecules were finally resolved by SDS-PAGE and detected with an anti-T7

monoclonal antibody (Novagen) used in conjunction with the ECL system (Pharmacia).

Binding efficiency was quantified by an imagine analysis program (Scionimagin).

Results


The C-terminal Stirrup ofArabidopsis TBP is Required for Binding to TFIIB

To test the role of the C-terminal stirrup domain of plant TBP in binding TFIIB,

single or double substitution mutations E144R, E146R and E144R/E146R of AtTBP2

were constructed and then evaluated in binding studies with AtTFIIB in vitro. Protein-

protein interactions in the absence ofDNA (TATAA element) were determined using

GST pull-down assays. Binding was quantified using an image analysis program

(Scionimage) with images scanned from X-ray film. In Fig. 2-4A, free TFIIB is shown

to bind immobilized GST-TBP and its mutants, indicating that TBP/TFIIB interaction

does not require pre-association of TBP with the TATAA motif. The binding efficiency

of TBP was significantly reduced by the C-terminal stirrup mutations (Fig. 2-4A). The

two single mutations, E144R and E146R, exhibited an approximately 50% reduction in

binding, while the double mutation E144R/E146R showed more than an 8-fold

reduction (Fig. 2-4B). During in vitro translation, synthesis of a C-terminal deletion

mutant of TFIIB was also evident, as indicated by the faster migrating band in Fig. 2-

4A. Although the amount of input protein for the truncated TFIIB was almost equal to

that of the wild type protein, this deletion mutant bound to TBP and its mutants with













A. Immobilized GST-TBP; TFIIB free

10% input


T7-AtTFHB --
T7-AtTFIIBAC --P


- o w


E144R E146R

GST-AtTBP2


E144R
E146R


N WT
O AC


Bound


WT


E144R


E146R E144R
E146R


GST-AtTBP2

Figure 2-4. Binding of free TFIIB to immobilized GST-TBP2 or its mutants.
(A) Western blot of bound TFIIB. T7-AtTFIIB was synthesized by a coupled
transcription/translation reaction in wheat germ. T7-AtTFIIBAC represents
prematurely terminated protein resulting in an undefined C-terminal truncation.
Bands were visualized by probing the western blot with anti-T7 monoclonal
antibody. (B) Quantification of imagines for (A). Bands on X-ray film were
scanned and then analyzed by computer using Scionimage software. Area units
are arbitrary.


10% input
0011


30.0

25.0

20.0

15.0

10.0

5.0


0.0 L


T7-TFIIB








about 50% efficiency (Fig. 2-4B). Similarly, a human TFIIB deletion mutant showed a

40% efficiency in binding with TBP when its C-terminal 30 or 80 residues were

truncated (Ha et al., 1993).

In a reciprocal experiment (Fig. 2-5) where GST-TFIIB was immobilized on

beads and the TBP is free in solution, similar results were obtained: C-terminal

mutations of TBP interfered TBP/TFIIB interactions. Both wild type and mutants of

TBP did not bind to GST control (data not shown), but bound to GST-TFIIB (Fig. 2-5).

Binding of TBP to GST-TFIIB was inhibited by TBP C-terminal stirrup mutations.

Inhibition by TBP mutant E146R remained at about 50%; however, inhibition by the

E144R mutation was more severe when TFIIB was immobilized, with a 7-fold reduction

seen in binding. Inhibition by the double TBP mutation (E144R/E146R) in this

experiment was more than 8-fold, which was almost equal to that obtained in the

reciprocal configuration shown in Fig. 2-4. Although mutation of the two glutamic acid

residues in the C-terminal stirrup ofarabidopsis TBP2 did not completely abolish

interactions with TFIIB, it is clear that mutation of these residues is severely disruptive

to binding, as is the case with human and yeast proteins (Lee and Struhl, 1997; Tang et

al., 1996).

The TBP-TFIIB Interaction is Dispensable in Basal Transcription in vivo

Basal transcription in maize suspension cells was determined using the GUS

reporter gene under control of the CaMV 35S minimal promoter (nt -46) which contains

no activation elements upstream of the TATAA motif (minimal promoter) (Odell et al.,

1985). The same reporter with the promoter deleted served as the negative control for no

transcription (promoterless/GUS). Experiments with the promoterless/GUS reporter














A. Immobilized GST-TFIIB; TBP free


10% Inputs


Binding to GST-AtTFIIB


T7-AtTBP2
& mutants


WT E144R E146R E144R WT E144R E146R E144R
E146R E146R


Bound

IT
WT


E146R


E144R


AtTBP2 and mutants (free)


Figure 2-5. Binding of free TBP to immobilized GST-TFIIB. (A) Western blot
of AtTBP2 and mutants bound to immobilized GST-AtTFIIB. Western blot was
probed with anti-T7 monoclonal antibody. T7-AtTBP2 and mutant proteins were
synthesized by a coupled transcription/translation reaction in rabbit reticulocyte.
(B) Quantification of imagines for (A) as discussed for Fig. 2-4.


E144R


B.


25.0


20.0


15.0


10.0


5.0


0.0


E146R


E144R


10% inputs


E144R
E146R






57

indicated that background levels of GUS activity were very low in the Black Mexican

Sweet cell line (column 1, Fig. 2-6). Using the empty vector as the effector, GUS

activity with the CaMV 35S minimal promoter was 12-fold higher than the

promoterless/GUS background (compare column 2 vs. 1, Fig. 2-6). This activity

represents basal transcription in maize cells, since the minimal promoter lacked

upstream elements.

Coexpression of the wild-type AtTBP2 effectorr) with the minimal

promoter/GUS reporter stimulated GUS activity 4-fold above levels obtained with the

reporter alone (compare column 4 vs. 2, Fig. 2-6). This result not only indicates that

arabidopsis TBP is compatible with the maize transcription machinery, but that the

elevation in TBP concentration was able to partially compensate for the lack of upstream

elements in the promoter. However, coexpression of AtTFIIB showed no effect on the

transcription (compare column 3 vs. 2, Fig. 2-6), suggesting that TFIIB is not rate

limiting for basal transcription in maize cells.

Next, the functional significance of the TBP-TFIIB interaction was examined in

basal transcription. If the TBP-TFIIB interaction is critical to basal transcription in vivo,

coexpression of the stirrup mutants of TBP with the minimal promoter/GUS reporter

should severely inhibit the basal transcription by competing for TATA binding with the

endogenous TBP. However, over-expression of mutant AtTBP2 with either single

(E144R, E146R), double (E144R/E146R), or triple (E144R/E146R/K197E) point

mutations enhanced basal transcription almost as efficiently as the wild-type TBP

(compare columns 5, 6, 7, and 8 vs. 4, Fig. 2-6). This lack of suppression by stirrup

mutations in TBP suggests that under these conditions of TBP over-expression,











Basal transcription in maize cells


TBP mutants


TBP2


E146R


E144R


4.3 -

4.0

3.5

3.0

2.5 -

2.0 -

1.5

1.0 -

0.5 -

0.0


T7
sector TFIIB
vector


ATATA


Figure 2-6. Effects of coexpression of TBP and TFIIB on basal
transcription in maize cells. Basal transcription of the GUS reporter was
driven by the CaMV 35S minimal promoter (nt -46) without upstream
activation elements. The same amount of DNA (10 jIg) was used for each
effector construct. The T7 vector was used to express TBP or TFIIB, and
contains the T7 epitope coding sequence. The construct with the minimal
promoter deleted (ATATA) served as a control for no transcription, and was
coexpressed with the T7 vector. Transcriptional activity was normalized by
the internal control LUC activity and expressed as arbitrary units of relative
GUS/LUC (nM Mu/cpm). The results of one representative experiment are
shown. The data for each treatment was the average of three replicates.


I
E144R
E146R
K197E


1 2 3 4 5 6 7 8
Effector constructs coexpressed with the basal transcription
reporter (CaMV 35S minimal promoter/GUS)






59

TBP/TFIIB interactions only play a minor role, if any, in supporting basal transcription.

The stimulation of basal transcription by over-expression of TBP argues that the

arabidopsis TBP was both expressed and functional for wild type and mutant forms of

the protein.

The TBP-TFIIB Interaction is Required for Activated Transcription by the GAL4
Promoter

To address whether the TBP-TFIIB interaction is important for the activated

transcription, experiments were conducted using the GAL4-driven reporter system

(GAL4xlO/GUS). The activator proteins were heterologous fusions between the Gal4

DBD and several different types of activation domains including the acidic activation

domains VP16 (mammalian virus) and GAL4 (yeast), the glutamine-rich activation

domain of ftzQ (Drosophila), and two activation domains from plant transcription

activators, VP1 (maize) and LpHSF8 (tomato heat shock factor). In separate

experiments, these activators from either plants or the more widely diverging organisms

were determined to function in the maize Black Mexican Sweet cell line, showing strong

activation of transcription when compared to Gal4 DBD alone (data not shown).

Transcriptional activity of these activation domains was then monitored by coexpression

of TBP, or its C-terminal stirrup mutants, with each activator in five parallel

experiments shown in Figs. 2-7 through 2-11. Coexpression of wild type TBP slightly

inhibited activity of ftzQ (compare column 2 vs. 1, Fig. 2-7), VP16 (compare column 2

vs. 1, Fig. 2-8) and VP1 (compare column 2 vs. 1, Fig. 2-11) by approximately 25%, but

enhanced Gal4 AD (compare column 2 vs. 1, Fig. 2-9) and LpHSF8 (compare column 2

vs. 1, Fig. 2-10) activities by more than 60%. Compared to wild type TBP, the E144R

mutation showed variable levels of enhancement for all five activation domains















Transcription activated by Gal4 DBD/ftzQ




TBP mutants


E144R


T7
vector


TBP


I
*
I
I
I
I
I
I
I
I
.
I
I
2



I
I
I


E146R


E144R
E146R


I I
1 2 3 4 5 6

Effector constructs coexpressed with Gai4 DBD/ftzQ


Figure 2-7. Effects of coexpression of TBP or the C-terminal stirrup
mutants on ftzQ transcriptional activity. Transcription of the GUS gene
was activated by Gal4 DBD/ftzQ bound to the GAL4 sites (10x)
upstream of the CaMV 35S minimal promoter (-46). The same amount
of DNA (10 pg) was used for co-expression of each effector construct.
Transcriptional activity was normalized by the internal control LUC
activity and expressed as arbitrary units of relative GUS/LUC (nM
Mu/cpm). The results of one representative experiment are shown. The
data for each treatment was the average of three replicates.


375




300


225


150




75-




0-


E144R
E146R
K197E
















Transcription activated by Gal4 DBD/VP16


TBP mutants


E146R


1 2 3


E144R
E146R


E144R
E146R
K197E


5 6


Effector constructs coexpressed with Gal4 DBD/VP16


Figure 2-8. Effects of coexpression of TBP or the C-terminal stirrup mutants
on VP16 transcriptional activity. The details are the same as in Fig. 2-7.


1500-


1250-


1000-


750-


500-


250-


0 1-















Transcription activated by Gal4 DBD/Gal4 AD




TBP mutants


T


T7
vector


I
I


BP
I
LI
|
I
I
I
I


I
I
I
I
I
2.


E144R


F


E146R


E144R
E146R

T


E144R
E146R
K197E


Effector constructs coexpressed with Gal4 DBD/Gal4 AD


Figure 2-9. Effects of coexpression of TBP or the C-terminal stirrup mutants
on Gal4 AD transcriptional activity. The details are the same as in Fig. 2-7.


400 -



320



240 -


IOU -



80-



S-













Transcription activated by Gal4 DBD/LpHSF8



TBP mutants
I I
500 -
E144R


400 TBP2


300 T7
vector


200

E146R E144R
100 E144R E146R
E146R K197E

0
1 2 3 4 5 6
Effector constructs coexpressed with Gal4 DBD/LpHSF8


Figure 2-10. Effects of coexpression of TBP or the C-terminal
stirrup mutants on LpHSF8 AD transcriptional activity. The details
are the same as in Fig. 2-7.






















Transcription activated by Gal4 DBD/VP1


TBP mutants


E146R


I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I


5 6
5 6


Effector constructs coexpressed with Gal4 DBD/VP1




Figure 2-11. Effects of coexpression of TBP or the C-terminal stirrup

mutants on VP1 transcriptional activity. The details are the same as in

Fig. 2-7.


T7

vector


300




250




200




150


E144R


TBP2


E144R

E146R


0 L


E144R

E146R

K197E






65

(compare column 3 vs. 2, Figs. 2-7 to 2-11) with ftzQ being the highest (compare

column 3 vs. 2, Fig. 2-7). Although the enhancement in activity is unexplained, a

minimal interpretation is that the E144R mutation of TBP did not impair activated

transcription in vivo.

In contrast to the stimulation in activity obtained with mutant E144R, the E146R

mutation significantly inhibited transcriptional activity for all five activators tested, with

the degree of reduction ranging from more than 2- to about 5-fold (compare column 4

vs. 2, Figs. 2-7 to 2-11). The double mutation E144R/E146R showed a more severe

inhibition ranging from 5- to 16-fold (compare column 5 vs. 2, Figs. 2-7 to 2-11).

Although the E144R mutation alone appeared to stimulate activity, when combined with

E146R (E146RE 144R) transcriptional activity was further reduced from that obtained

from E146R alone. A third site of charge-charge interaction between TBP and TFIIB

involves K197 near the C-terminus of TBP (Nikolov et al., 1995). However, the triple

mutation, E144R/E146R/K197E, showed no further inhibition of transcription compared

to E144R/E146R (compare column 6 vs. 5, Figs. 2-7 to 2-11), suggesting that double

mutation of the stirrup of TBP is sufficient to abolish the TBP-TFIIB interaction in vivo.

Taken together, these results strongly contrast those observed in basal transcription, and

indicate that activated transcription driven by the synthetic GAL4 promoter is highly

dependent on the TBP-TFIIB interaction in vivo.

The inhibition caused by coexpression of TBP with VP1, VP16, and ftzQ may be

due to transcriptional squelching defined in an early study (Gill and Ptashne, 1988). In

the TBP titration experiment depicted in Fig. 2-12, ftzQ activity was first enhanced with

low amounts of TBP expression vector and then suppressed when levels of the TBP
















CaMV 35S


400


300



200


)0 .o



0 2.5 5 10


AtTBP2


--






0 2.5 5 10 pg DNA

AtTBP2


Figure 2-12. The effects of TBP concentration on transcriptional activation
in maize cells. Different amounts of T7-AtTBP2 DNA were coexpressed
with either CaMV 35S/GUS reporter whose activity is endogenous to maize
cells, or the GAL4/GUS reporter activated by Gal4 DBD/ftzQ. Total
amounts of DNAs were kept constant by addition of the T7 vector
expressing only the T7-epitope. GUS activity was normalized by the internal
control LUC activity and expressed as arbitrary units of relative nM Mu/cpm.
The results of one representative experiment are shown. The data for each
treatment was the average of three replicates.


Gal4 DBD/ftzQ






67

were further increased (compare columns 6, 7, and 8 vs. 5, Fig. 2-12), implying that ftzQ

activity was squelched by high levels of TBP protein. However, this squelching effect

was not observed in transcription driven by the CaMV 35S promoter (compare columns

2, 3, and 4 vs. 1, Fig. 2-12), suggesting that optimal TBP concentrations vary between

different activators. This activator-dependent squelching by TBP is also observed in

other systems in vivo (Sadovsky et al., 1995). In general, the degree of squelching seen

in these experiments was not severe and did not alter conclusions regarding the

importance of TBP-TFIIB interaction in activated transcription.

The degree of transcriptional suppression by stirrup mutations of TBP varied

between the activation domains tested. For example, with the double mutation

E144R/E146R, transcription was reduced 16-fold for ftzQ (compare column 5 vs. 2, Fig.

2-7), 12-fold for VP16 (compare column 5 vs. 2, Fig. 2-8), 11-fold for LpHSF8

(compare column 5 vs. 2, Fig. 2-10), 9-fold for VP1 (compare column 5 vs. 2, Fig. 2-

11), but only 4.5-fold for GAL4 (compare column 5 vs. 2, Fig. 2-9). A nearly 4-fold

difference in the degree of suppression was seen, with ftzQ being the most sensitive and

Gal4 AD the least affected.

Requirement for TBP-TFIIB Interaction Confirmed Using an Altered Specificity TBP

A TBP altered-specificity system was employed to further demonstrate the effect

of TBP stirrup mutations on activated transcription, free of possible interference by

endogenous TBP. The TATAA element of the GAL4/GUS reporter was mutated to

TGTAA so that activity would be dependent on expressed TBPm3, which is able to

recognize TGTAA in addition to TATAA (Strubin and Struhl, 1992). To demonstrate

the lack of recognition of the TGTAA motif by endogenous maize TBP, the empty






68

vector (T7 vector) was coexpressed with the TATAA and TGTAA GUS reporters as

shown in columns 1 and 2 of Figs. 2-13 through 2-15. Endogenous activity was much

reduced with the TGTAA reporter. Furthermore, over-expression of wild type

arabidopsis TBP produced only low increases in activity compared to the endogenous

TBP, which is seen by comparing columns 2 and 3 of Figs. 2-13 through 2-15. In

contrast, over-expression ofAtTBPm3 resulted in significantly more activity compared

to wild type AtTBP, ranging from a very modest increase in activity for VPI (Fig. 2-14;

column 4) to a complete restoration of endogenous activity for the tomato heat shock

factor LpHSF8 (Fig. 2-15A; column 4).

The importance of TBP-TFIIB interactions was demonstrated by analysis of the

C-terminal stirrup mutations introduced into the altered specificity system. Mutations

E144R, E146R and E144R/E146R were introduced into AtTBPm3 and assayed using

the TGTAA reporter. When activated transcription relied on exogenous TBPm3, all the

three stirrup mutations suppressed activated transcription (Figs. 2-13 through 2-15).

Unlike in the stimulation seen with TBP and the TATAA reporter, the E144R mutation

inhibited activity by 2-fold for both ftzQ (compare column 5 vs. 4, Fig. 2-13) and VPI

(column 5 vs. 4, Fig. 2-14), and 3-fold for LpHSF8 (column 5 vs. 4, Fig. 2-15A).

Consistent with the results obtained with TBP, with TBPm3 the E146R mutation also

showed more severe inhibition than E144R, with reductions in activity ranging from 6-

fold for ftzQ (compare column 6 vs. 4 and 5, Fig. 2-13) to 20-fold for LpHSF8 (column

6 vs. 4 and 5, Fig. 2-15A). The double mutation E144R/E146R, again, showed the

strongest inhibition for all the three activation domains, exhibiting up to a 55-fold

reduction in activity for LpHSF8 (compare column 7 vs. 4, Fig. 2-15A). Inhibition of











Gal4 DBD/ftzQ activity in TGTA/TBPm3
coupled transcription system


70 T7
vector
60 TBPm3
wt
S50

0 40

30 E144R

20 TBP
T7
7 |E146R E144R
10 vector E146R

0 1 I I --i a T
1 2 3 4 5 6 7
TATA I I
reporter TGTA reporter


Figure 2-13. Effects of TBPm3 and its C-terminal stirrup mutants on ftzQ
transcriptional activity in the TGTA/TBPm3 coupled system in maize cells.
AtTBP2 was mutated by three amino acid substitutions within its concave
DNA binding surface to generate TBPm3 (Strubin and Struhl, 1992). In the
context of this TBPm3, single, double, or triple point-mutation was
introduced into the C-terminal stirrup as labeled in the figure. The same
amounts of different effector plasmids were coexpressed with the reporter
containing the mutated TATA motif, TGTA. Column 1 was the control for
normal ftzQ activity using the wild type TATA reporter. ftzQ activity was
expressed as relative GUS/LUC activity. The results of one representative
experiment with three replicates are shown. The data for each treatment was
the average of three replicates.

















Gal4 DBD/VP1 activity in TGTA/TBPm3
coupled transcription system











TBPm3


E144R
E146R E146R

6 7
6 7


TGTA reporter


Figure 2-14. Effects of TBPm3 and its C-terminal stirrup mutants on
VPI transcriptional activity in the TGTA/TBPm3 coupled system in
maize cells. The details are the same as in Fig. 2-13.


90

80


T7
vector


TATA
reporter
















Gal4 DBD/LpHSF8 activity in TGTA/TBPm3
coupled transcription system


TBPm3


T7
vector

T


T7
vector



2


TBP

T


E144R


E144R
E146R E14
E146R
6 7-
6 7


TATA
reporter


TGTA reporter


Figure 2-15. Effects of TBPm3 and its C-terminal stirrup mutants on
LpHSF8 transcriptional activity in the TGTA/TBPm3 coupled system in
maize cells. (A) Relative GUS/LUC activity of transcription activated
by LpHSF8 activation domain. The details are the same as in Fig. 2-13.


490

420

350-

280

210-

140

70

0-


1


















400000
T7
350000 vector


300000


250000


200000

150000


100000

50000


0


1
TATA
reporter


2
I


TBPm3


wt



TBP








3 4


TGTA reporter


Figure 2-15 -- continued
(B) The same as in (A) except that GUS activity was not normalized by
luciferase activity of the internal control.


E144R


E146R E144R
E146R


5 6 7






73

transcription by the C-terminal stirrup mutations was also apparent even if GUS activity

was not normalized by the internal control luciferase activity (Fig. 2-15B). Overall,

these results are parallel to those observed in experiments using the TATAA/TBP

system, although it appears that TBPm3 is much more sensitive to C-terminal stirrup

mutations than TBP. In both systems, residue E146 of TBP appears to be more critical

than E144 in supporting the TBP-TFIIB interaction in vivo, which is consistent with

predictions based on structure (Nikolov et al., 1995).

Complex Promoters Show Much Less Dependence on the TBP-TFIIB Interaction

In addition to testing the synthetic GAL4 promoter driven by a single activation

domain fused to the Gal4 DBD, two natural promoters, the CaMV 35S and maize

ubiquitin promoters, were also evaluated regarding the significance of the TBP-TFIIB

interaction. These two promoters are more complex than the synthetic GAL4 promoter

and rely on multiple activator proteins endogenous to maize cells for activity (Benfey et

al., 1990; Genschik et al., 1994; Hoffman et al., 1991; Lam et al., 1989). The CaMV

35S/GUS and Ubi/LUC reporters were coexpressed with either TBP or its mutants in the

same experiment. The effects of TBP mutations on CaMV 35S and ubiquitin promoter

activities were presented in Fig. 2-16 and Fig. 2-17, respectively. Transcription by the

CaMV 35S promoter in transient assays was strongly stimulated by over-expression of

wild-type TBP and all stirrup mutants (compare columns 2, 3, 4, 5, and 6 vs. 1, Fig. 2-

16A and B), while ubiquitin promoter activity was only minimally affected by

coexpression of TBP and mutants (compare columns 2, 3, 4, 5, and 6 vs. 1, Fig. 2-17). In

contrast to the strongly inhibitory effect of stirrup mutations in transcription driven by

the GAL4 promoter, transcription of these two natural promoters was almost completely

















Transcription activated by wild-type
CaMV 35S promoter in maize cells



TBP mutants


E144R


TBP


T7
vector

1 2


E144R
E146R
E146R


E144R
E146R
K197E


4 5 6


Effector constructs coexpressed
with the CaMV 35S/GUS reporter


Figure 2-16. Effects of TBP and C-terminal stirrup mutants on
CaMV 35S activity in maize cells. (A) Different effectors with the
same amount of DNA were coexpressed with the CaMV 35S/GUS
reporter. CaMV 35S promoter activity in maize cells was expressed
as arbitrary units of relative GUS/LUC (nM Mu/cpm). The results of
one representative experiment with three replicates are shown.


150 -

120 -

90 -

60 -

30 -
a -













160000 TBP mutants
E144R
140000


120000


100000 E146R

80000 E1441


60000


40000


T7
20000 'vector


0 -
1


TBP



r'


E146R


E144R
E146R
K197E

T


R


Effector constructs coexpressed
with the CaMV 35S/GUS reporter
Figure 2-16 -- continued
(B) The same as in (A) except that GUS activity was not normalized
by luciferase activity of the internal control.
















Transcription activated by ubiquitin
promoter in maize cells




AtTBP2 mutants


T7
vector


E144R
E146R E146R


"-r TBP E144R


1 2 3 4


E144R
E146R
K197E
T


5 6


Effector constructs coexpressed with the Ubi/LUC reporter



Figure 2-17. Effects of TBP and C-terminal stirrup mutants on
ubiquitin promoter activity in maize cells. Different effectors with
the same amount of DNA were coexpressed with the Ubi/LUC
reporter. Ubiquitin promoter activity in maize cells was expressed
as counts per minute (cpm) of photon emission. The results of one
representative experiment with three replicates are shown.


200

150

100

50


0 L-








insensitive to the same TBP mutations. Compared to a simple promoter such as GAL4,

serving as the binding site for a single type of activator, complex natural promoters are

able to minimize the requirement for direct TBP-TFIIB interaction, perhaps due to the

utilization of multiple recruitment pathways made possible by the involvement of

multiple activators.

These results also indicate that TBP was a rate-limiting factor for transcription

driven by the CaMV 35S promoter, but not for the ubiquitin promoter. Similar

differential responses by different activators to over-expression of TBP were previously

observed in HeLa (Sadovsky et al., 1995). The increases in activity of the ubiquitin

promoter seen with the mutant forms of TBP, compared to exogenous wild type TBP,

may indicate that the mutated TBPs are less efficient, but still functional (Fig. 2-17).

This decrease in efficiency is presumably reflected in their reduced capacity to squelch.

A similar effect was seen with the CaMV 35S promoter, where most of the stirrup

mutants showed less activity than exogenous wild type TBP, but nevertheless stimulated

transcription compared to endogenous TBP (T7 vector).

Reliance on Multiple Activation Pathways Can Partially Compensate Suppression by
TBP Stirrup Mutations

To test whether the TBP-TFIIB interaction is still important in transcription

involving multiple recruitment pathways, two activation domains were fused together to

drive GAL4-dependent transcription. The Gal4 AD was fused to two different activation

domains to produce two double activators, Gal4 DBD/VP6-Gal4 AD and Gal4

DBD/ftzQ-Gal4 AD. These were coexpressed in transient assays with wild type or

mutated TBP to evaluate suppression by stirrup mutants. As seen in Fig. 2-18,

transcription activated by ftzQ-Gal4 AD remained sensitive to the TBP stirrup















ftzO


T7 TBP
vector rL
T l-


GAL4-dependent transcription


-Gal4 AD VP1


T7
vector TBP














E146R
I














E144R
E146R


i l


1 2 3 4


16-Gal4 AD








E146R
-r


7 8


Effector constructs coexpressed with either ftzQ-
Gal4 AD or VP16-Gal4 AD double activators

Figure 2-18. Double activators show variable dependence on TBP-TFIIB
interactions. Gal4 DBD/ftzQ-Gal4 AD (left panel) or Gal4 DBD/VPI6-Gal4 AD
(right panel) double activators were coexpressed in transient assays with the
GAL4/GUS reporter and wild-type AtTBP2 or the stirrup mutants. T7 vector
represents the empty vector control utilizing endogenous TBP. The results of one
representative experiment with three replicates are shown.


1200


900 -


600




300


0 1






79

mutations, whereas, that driven by VP16-Gal4 AD was less susceptible. The double

mutation E144R/E146R showed an 80% inhibition for the activator ftzQ-Gal4 AD

(compare column 4 vs. 2, Fig. 2-18), but only a 45% reduction in transcription activated

by VPl6-Gal4 AD (compare column 8 vs. 6, Fig. 2-18). The differential effects of

stirrup mutants on these two double activation domains reveal an unexpected complexity

in mechanism that must underlie these results. On one hand, the decreased sensitivity to

TBP stirrup mutations exhibited by VP16-Gal4 AD suggests that the presence of

multiple pathways (or contacts) of activation inherent in double activation domains leads

to a diminished dependence on TBP-TFIIB interactions for activated transcription.

However, for ftzQ-Gal4 AD, this model is contradicted since very little decrease in

reliance on TBP-TFIIB interactions was evident. Clearly, the importance of direct TBP-

TFIIB interactions in activated transcription is context sensitive, for reasons that are

poorly understood.

Discussion

The present study is the first in plants to examine the biological function of an

important protein-protein interaction within the PIC, contacts between TBP and TFIIB.

Transient assays in maize cells demonstrated that the TBP-TFIIB interaction can be a

major pathway for activated transcription; however, the functional importance of this

association to the process of transcription is clearly context sensitive in plants. It is

essential for efficient gene expression in certain situations, and in others, contacts

between these two general transcription factors appear to be dispensable. Activated

transcription from a simple promoter driven by a single activation motif is heavily

dependent upon the TBP-TFIIB interaction, whereas in complex natural promoters, the






80

same interaction make only minor contributions to overall activity. Likewise,

interactions between TBP and TFIIB were not important in basal transcription from the

minimal CaMV 35S promoter. Although the same domains of TBP participate in

binding TFIIB in both human and plants, the relative contributions of individual residues

show clear distinctions between these organisms.

Using GST pull-down assays, direct protein-protein interaction between AtTBP2

and AtTFIIB was observed in vitro. The present experiments differ from many of the

previous studies by examining TBP-TFIIB interactions in the absence of TATAA

element DNA, which is commonly used in employing EMSAs (Chou and Struhl, 1997;

Lee and Struhl, 1997; Tang et al., 1996). This work, and a previous study using human

components (Ha et al, 1993), indicates that the association of TBP with DNA is not a

prerequisite for the association of TBP with TFIIB. In addition, the reduction in binding

of the inadvertent C-terminal deletion of TFIIB (Fig. 2-4) and the clear involvement of

the C-terminal stirrup of TBP in the TBP-TFIIB interaction are consistent with previous

studies in humans indicating that these domains are active in binding (Ha et al, 1993;

Hisatake et al., 1993).

Although the TBP-TFIIB interaction appears to also involve residues of the C-

terminal stirrup of TBP in plants, there are some significant differences between plants

and other organisms. In humans, TBP stirrup residues E284 and E286 (corresponding to

E144 and E146 in AtTBP2) are both critically important in binding with hTFIIB.

Mutations E284A and E286A showed more than 20-fold inhibition in binding (Tang et

al, 1996). Likewise, the same C-terminal stirrup mutations eliminated the binding for

yeast proteins in similar experiments (Lee and Struhl, 1997). Compared to human and








yeast systems, analogous point mutations are much less severe in plants, ranging from

only 2- to 7-fold in the GST pull-down assays. It is not known whether this difference

may be due to differences in the method of assay. For example, is the nature of

association of TBP with DNA in EMSA assays the one that further enhances the

dependence of the TBP-TFIIB interaction on these two glutamic acid residues? Another

consideration is the structural differences between plant and animal TBPs. Compared to

human and yeast TBPs, AtTBP2 lacks the nonconserved N-terminal region whose

function is still unclear with respect to transcriptional regulation.

In maize suspension cells, basal transcription dependent on the CaMV 35S

minimal promoter was specifically stimulated by coexpression ofTBP but not TFIIB.

Although basal transcription is normally defined by in vitro experiments using

reconstituted transcription systems, it can occasionally be observed in vivo. For

example, expression of basal transcription in Drosophila L2 cells has been reported

(Colgan et al., 1993). This observation in Drosophila and the results obtained in this

study indicate that basal transcription can also occur in vivo. There are other parallels

between the present study in plants and the Drosophila study. For example, basal

transcription in Drosophila was stimulated by coexpression ofdTBP by 20-fold,

whereas dTFIIB was totally ineffective in stimulating basal activity (Colgan et al.,

1993). Although AtTFIIB protein expression in maize cells was not determined in the

present study, it seems to be normal, since preliminary experiments showed that

activated transcription by several GAL4 fused activation domains was stimulated by

coexpression of AtTFIIB (not included in this dissertation). Therefore, it is likely that

the TBP or TFIID concentration on the promoter, rather than TFIIB or holoenzyme, is






82

limiting for basal transcription in vivo. This implies that recognition of the TATA motif

by TBP or TFIID may be the most important step determining levels of basal

transcription.

In contrast to the in vitro system, basal transcription in maize cells appears not to

require the TBP-TFIIB interaction. This conclusion is evident since AtTBP2 mutations

at the C-terminal stirrup did not interfere with the positive function of TBP in basal

transcription. Stimulation of this in vivo basal activity by exogenous TBP was

comparable between the wild type and the mutant proteins as shown in Fig. 2-6.

However, reconstituted in vitro transcription using human components indicates that a

single point mutation in the C-terminal stirrup of hTBP is sufficient to abolish basal

transcription (Bryant et al, 1996). These opposing results indicate that fundamental

differences may exist in the properties of basal transcription between plants and humans

or, alternatively, between in vivo and in vitro transcription systems. Regardless of the

nature of differences with other organisms, it is apparent from these results that an

alternative interaction pathway is available in plant cells for the PIC assembly to support

basal transcription when the TBP-TFIIB interaction is disrupted.

The TBP-TFIIB interaction is critical for function when a synthetic GAL4

promoter is activated by a single type of activation domain. In this simple experimental

system, either holoenzyme or TFIID is initially recruited to the promoter. Once this

occurs, the rate-limiting step is likely in the subsequent recruitment of the second

complex (Gonzalez-Couto et al, 1997). Under these conditions, the strength of

interaction between TFIID and holoenzyme (TFIIB) may then be correlated with the

level of transcriptional activity. Although many interactions among general transcription






83

factors documented in vitro could serve this bridging function, the TBP-TFIIB

association clearly plays a major role in joining the two complexes during

transcriptional activation in plant cells. This conclusion is based on the TATAA-TBP

and TGTAA-TBPm3 coupled transcription systems in which activated transcription was

most severely inhibited by the E144R/E1 46R mutation of AtTBP2 for all five activation

domains tested.

The roles of the two individual glutamic acids in the C-terminal stirrup of TBP

appear to differ between in vivo and in vitro transcription systems. Residue E144 was

equally, or probably more, important than E146 in binding TFIIB in vitro as shown in

Figs. 2-4 and 2-5. However, in activated transcription in vivo using an assortment of

activation motifs, E146 was much more critical than E144 (Figs. 2-7 through 2-11, and

2-13 through 2-15). It seems that the degree of involvement in TFIIB binding in vitro

does not strictly correlate with the importance of a particular residue in supporting

activated transcription in vivo. Unlike the direct interaction in vitro in an isolated

system, the in vivo TBP-TFIIB interaction takes place in the context of the PIC in

association with the TATAA motif of the promoter. This interaction is probably much

more complicated than that observed in vitro in terms of possible influences from other

factors including the TATAA, the PIC components, and the activator. In fact, in the

crystal structure of the TATAA-AtTBP2-hTFIIBc ternary complex, E146 is apparently

more involved in TFIIB binding than E144 (Nikolov et al, 1995). Therefore, it is

possible that the relative strength of these two residues in TFIIB binding is different in

the presence and absence of the TATAA motif. Indeed, TBP shows some

conformational distortion after binding to the DNA (Kim and Burley, 1994). This






84

conformational change may be an important parameter in determining the relative

importance of E144 and E146 in TFIIB binding. In this regard, the in vivo results

showing E146 to be more important than E144 in supporting activated transcription is

supported by the structural data (Nikolov et al, 1995), but not in good agreement with

the in vitro GST pull-down results, perhaps due to the lack of DNA in the in vitro

complex.

The functional importance of the TBP-TFIIB interaction in supporting activated

transcription in vivo varies between plants, humans and yeast. Transcription activated by

several different activation domains, including VP16, is completely independent of the

TBP-TFIIB interaction in yeast cells (Chou and Struhl, 1997; Lee and Struhl, 1997), but

totally dependent on this connection in HeLa cells (Tansey and Herr, 1997). Single

mutations in either of the two glutamic acid residues totally eliminate VP16 activity in

the TGTAA/TBPm3 coupled system in HeLa cells (Tansey and Herr, 1997), suggesting

that both residues are absolutely required in activated transcription. In plants, however,

these two residues appear to be partially redundant in function, showing proportional

inhibition on transcription by single and double mutations using an analogous

TGTAA/TBPm3 coupled system (Figs. 2-13 through 2-15). Therefore, the relative

importance of E144 and E146 of TBP in living cells apparently differs between plants

and humans, although transcriptional activation in both systems requires direct contact

between TBP and TFIIB. In this respect, the pattern of dependence of activated

transcription on the TBP-TFIIB interaction in vivo exhibited by plant transcription

seems to position plants closer to humans than to yeast.








In contrast to the GAL4-dependent system, the importance of the TBP-TFIIB

interaction in activated transcription is not apparent with the natural complex promoters

such as the CaMV 35S and the ubiquitin promoters. For example, CaMV 35S promoter

activity was strongly stimulated by both wild type and mutant TBP proteins in the

coexpression assays (Fig. 2-16). Moreover, activity of the ubiquitin promoter (internal

control construct) was also not impaired by TBP mutations in these assays (Fig. 2-17).

Therefore, unlike the case with the simple synthetic GAL4 system, TBP stirrup mutants

are, surprisingly, not defective in transcription driven by either of the two natural

complex promoters.

The reduced requirement for a strong TBP-TFIIB interaction shown by the

CaMV 35S and ubiquitin promoters suggests that enhanced stability of the PIC may

result from the involvement of multiple types of activation domains bound to the

upstream elements. The wild type CaMV 35S and GAL4-dependent promoters only

differ in the types and configurations of upstream elements. The core promoter is

identical between the two reporters and is derived from the CaMV 35S minimal

promoter (nt -46). For the GAL4 promoter, transcription is dependent only on the GAL4

element, which in these experiments serves as the binding site for a single type of

activation domain per assay. However, the upstream region of the wild-type CaMV 35S

promoter contains multiple factor binding elements, potentially driven by multiple

activator proteins (Benfey et al., 1990; Lam et al., 1989). One consequence of the

involvement of multiple activator proteins is the possibility that multiple recruitment

pathways may be employed for general transcription factors. This involvement of

multiple interactions in recruitment may stabilize the PIC in a cooperative manner






86

compared to recruitment dependent on a single type of activation domain and lessen the

importance of any single interactions, including TBP-TFIIB contact. Impairment of one

interaction pathway, such as TBP-TFIIB contact, may be compensated by alternate

pathways present in complex natural promoters.

Consistent with this view, activator constructs containing the two activation

domains VP16 and the Gal4 AD were less affected by TBP stirrup mutants than the

corresponding single activation domains, showing only 45% inhibition with the double

mutation E144R/E146R (Fig. 2-18). However, this lessened dependence on the TBP-

TFIIB interaction by multiple activation motifs is not universal, since the ftzQ-Gal4 AD

double activator was still very sensitive to TBP stirrup mutations, with activity reduced

5-fold by the E144R/E146R mutation. Although it has been demonstrated that an

artificial activator comprised of two different activation motifs may show a reduced

requirement for the TBP-TFIIB interaction, unexpectedly, this dependence is also

specific to the particular combination of activation motifs employed.

An important assumption underlines much of the interpretation of results

presented in this study that the mutations introduced into the C-terminal stirrup of

AtTBP2 do not affect stability of the protein. Since the transiently expressed proteins

were not synthesized in sufficient amounts to be detectable by western blotting using

chemiluminescent visualization, the question remains. However, there are several lines

of indirect evidences supporting the assumption that the mutations did not affect TBP

levels in maize cells. First, basal transcription was stimulated by both wild type and

mutant TBP at similar levels (Fig. 2-6). Second, similar levels of stimulation between

wild type and mutant TBPs were observed for the CaMV 35S promoter (Fig. 2-16).






87

These two observations strongly argue that protein levels in maize cells were similar

between wild type and mutant TBP. The third line of reasoning draws on results from an

analogous system where these same mutations introduced into human TBP did not alter

TBP expression or stability in HeLa cells (Tansey and Herr, 1997). The dramatic

inhibitory effects of AtTBP2 mutations on activated transcription is, therefore, assumed

to reflect the critical roles of the C-terminal stirrup domain of TBP, and support the

conclusion that association between TBP and TFIIB can play a major role in PIC

assembly and activated transcription in plant cells.













CHAPTER 3
THE SPECIFIC INTERACTIONS WITH TBP AND TFIIB IN VITRO SUGGEST
14-3-3 PROTEINS MAY PARTICIPATE IN THE REGULATION OF
TRANSCRIPTION WHEN PART OF A DNA BINDING COMPLEX

Introduction


The 14-3-3 family of proteins was initially characterized by Moore and Perez

(1967) as acidic, soluble proteins which are highly abundant within bovine brain tissues.

Now approximately fifty 14-3-3 genes have been identified from animals, plants and

yeast by researchers in very different fields of study (Ferl, 1996; Wang and Shakes,

1996). In many cases, there are multiple 14-3-3 genes coding for different isoforms,

which are ubiquitously expressed in different cell types. As many as ten 14-3-3 isoforms

have been identified from arabidopsis (Wu et al., 1997b). Sequence comparison of 14-3-

3 cDNAs indicates that 14-3-3 proteins are highly conserved and widely distributed

phylogenetically (Wang and Shakes, 1996). X-ray crystallographic studies indicate

nearly identical structures between two isoforms, suggesting that all isoforms have very

similar structural features (Liu et al., 1995; Xiao et al., 1995a).

While the sequence and structure of the 14-3-3 protein family are highly

conserved, their biological functions are highly diversified in different systems. The 14-

3-3 proteins have been commonly recognized as regulators of a series of kinases

important in multiple signaling pathways, including Raf (Freed et al., 1994; Irie et al.,

1994; Li et al., 1995), Ras (Gelperin et al., 1995; Rommel et al., 1996), Bcr

(Braselmann and McCormick, 1995; Reuther et al., 1994) and PKC (Dellambra et al.,






89

1995; Toker et al., 1992). In animal brain cells, 14-3-3 proteins can activate

phosphorylated tyrosine tryptophan hydroxylase (TPHase), which is an important

enzyme involved in neural transmission (Ichimura et al., 1987; Yamauchi et al., 1981).

In permeabilized adrenal chromafm cells, 14-3-3 proteins are able to mediate calcium-

dependent exocytosis (Morgan and Burgoyne, 1992). In yeast, 14-3-3s are required for

cell viability (Van Heusden et al., 1994) and play a role in a mechanism that acts as

checkpoint for mitotic DNA damage repair (Ford et al., 1994). 14-3-3 proteins also

appear to have chaperone-like function since they can facilitate protein translocation

through the mitochondrial membrane by their ATPase activity (Alam et al., 1994). In

plants, 14-3-3 proteins are the receptor for the phytotoxin fusiccoccin (Marra et al.,

1994), and one isoform specifically inhibits nitrate reductase activity from spinach cells

(Bachmann et al., 1996). In several species which include dicots and monocots, 14-3-3

proteins are found in a sequence-specific DNA binding complex in vitro (Lu et al.,

1992; de Vetten et al., 1992) and are localized in the nucleus in vivo (Bihn et al., 1997).

Although the 14-3-3 protein family appears to be involved in a wide range of biological

functions, a common theme regarding function is their property for interactions with

other proteins (Ferl, 1996).

Biochemical characterizations of the 14-3-3 proteins have shown that they are

able to form homo- or heterodimers through their N-terminal dimerization domains

(Jones et al., 1995; Luo et al., 1995; Wu et al., 1997a). The 14-3-3 protein dimer is cup-

shaped containing a spacious internal area and negative residues located in the C-

terminal portion of each monomer. These charged residues align on the surface to form a

negative groove (Liu et al., 1995). This topological feature is predicted to be highly








conserved because the negative residues are conserved between the numerous isoforms.

This negative groove is thought to be a site for protein-protein interactions (Liu et al.,

1995). In recent studies, 14-3-3s were designated as "phosphoserine-binding proteins"

because many of their interacting proteins contain the consensus motif RSXpSXP and

require a phosphorylated serine residue for interactions to occur (Muslin et al., 1996).

The binding of a 14-3-3 protein results in inhibition of dephosphorylation of the

phosphoserine in the partner protein (Bachmann et al., 1996; Banik et al., 1997).

Phosphoserine binding was thought to serve as a general mechanism for 14-3-3 proteins

to participate in a variety of diverse cellular functions (Muslin et al., 1996). Domain

mapping for 14-3-3 proteins has identified a C-terminal region called box-1 which is

able to independently mediate phosphoserine binding (Ichimura et al., 1997). The

isolated box-1 domain can not form a dimer structure, yet still retains full capacity for

binding other proteins (Ichimura et al., 1997; Ichimura et al., 1995). Therefore, the

importance of the dimeric structure and the role of the negative groove of 14-3-3

proteins in protein-protein interactions remain unclear.

Although phosphoserine binding can explain most interactions involving 14-3-3

proteins, there are several exceptions. For example, the Cl domain of myosin II heavy-

chain-specific protein kinase C (MHC-PKC), which was shown to interact with 14-3-3

protein, does not contain the phosphoserine motif (Matto-Yelin et al., 1997). In the case

of the glucocorticoid receptor, the potential RSXpSXP motif is present only in its

activation domain, not in the ligand binding domain where 14-3-3 ir preferentially binds

(Wakui et al., 1997). These exceptions suggest that phosphoserine binding is but one of

several possible modes of interactions with 14-3-3 proteins.




Full Text
24
terminal stirrup of TBP. The El is a positive amphipathic helix, and the C-terminal
stirrup is negative in charge. It is likely that charge-charge interactions between the two
motifs account for the major binding energy. Amino acid substitution for either one of
the two glutamic acid residues at this stirrup totally abolished the formation of the
TATA-TBP-TFIIB complex in EMSA, demonstrating the critical roles of the two
negative residues in mediating the TBP-TFIIB interaction in both human and yeast (Lee
and Struhl 1997; Tang et al., 1996). However, since most of the studies on the TBP-
TFIIB interaction employed TBP prebound to DNA, it is not clear whether TBP with
DNA is the only conformation able to recognize TFIIB, or if the TBP-TFIIB interaction
can occur in the absence of DNA. Only one study used a pull-down assay to investigate
the direct interaction between hTBP and hTFIIB in the absence of DNA (Ha et al.,
1993). The results are in good agreement with the structural and EMSA data in that both
proteins used their C-terminal core domains to interact each other. Therefore, TFIIB is
able to recognize TBP with or without DNA.
The functional importance of the TBP-TFIIB interaction in transcription has
recently been tested by yeast genetic and the HeLa in vivo transcription system using a
sequential altered TATA-TBP-TFIIB specificity array. In yeast cells, the replacement of
the wild-type yTBP gene by its counterpart mutated in the C-terminal stirrup has little
effect on transcription by Gal4 and Gcn4 (Lee and Struhl, 1997). These TBP mutants do
not affect transcription driven by the pure recruitment of TFIIB using the fusion protein
LexA-TFIIB. Furthermore, in a system exhibiting pure recruitment of TBP, that is a
LexA-TBP fusion protein, these TBP mutants are functionally identical to the wild-type
protein in activating transcription from promoters containing the LexA binding site (Lee


GUS and Luciferase Assays 51
Protein Expression and Purification from E. coli 51
In vitro Protein Translation 52
In vitro GST Pull-Down Assay 52
Results 53
The C-terminal Stirrup of AtTBP2 is Required for Binding to AtTFIIB 53
The TBP-TFIIB Interaction is Dispensable in Basal Transcription in vivo 55
The TBP-TFIIB Interaction is Required for Activated Transcription by
the GAL4 Promoter 59
Requirement for TBP-TFIIB Interaction Confirmed Using an Altered
Specificity TBP 67
Complex Promoters Show Much Less Dependence on the TBP-TFIIB
Interaction 73
Reliance on Multiple Activation Pathways Can Partially Compensate
Suppression by TBP Stirrup Mutations 77
Discussion 79
THE SPECIFIC INTERACTIONS WITH TBP AND TFIIB IN VITRO
SUGGEST 14-3-3 PROTEINS MAY PARTICIPATE IN THE
REGULATION OF TRANSCRIPTION WHEN PART OF A DNA
BINDING COMPLEX 88
Introduction 88
Materials and Methods 92
Protein Expression in E. coli 92
Protein Purification from E. coli Lysate 93
In vitro Protein Translation 94
In vitro GST Pull-Down Assay 94
Site-Specific Point Mutagenesis 95
Transient Expression Assay 95
Results 96
Plant 14-3-3 Proteins Interact with Human TFIIB in vitro 96
Conserved C-terminal Core of hTFIIB Binds Arabidopsis 14-3-3 Protein 98
Arabidopsis 14-3-3 Protein and VP16 Show Similarities in Interactions
with hTFIIB 100
Human 14-3-3 o Shows Affinity for Human TFIIB, TBP, and TAFh32,
but not for TAFh55 104
Human 14-3-3 u Contains Two Domains That Bind TFIIB 105
Alanine Substitutions in 14-3-3 Helix 7 Identify Amino Acids Critical
for Binding TBP and TFIIB 105
At 14-3-3 Discussion 114


71
A.
Gal4 DBD/LpHSF8 activity in TGTA/TBPm3
coupled transcription system
o
D
_j
CO
=)
o
490 1
wt
420 T7 t
vector
12 3 4
TATA
TBPm3
E144R
reporter
TGTA reporter
E144R
E146R
7
Figure 2-15. Effects of TBPm3 and its C-terminal stirrup mutants on
LpHSF8 transcriptional activity in the TGTA/TBPm3 coupled system in
maize cells. (A) Relative GUS/LUC activity of transcription activated
by LpHSF8 activation domain. The details are the same as in Fig. 2-13.


76
Transcription activated by ubiquitin
promoter in maize cells
AtTBP2 mutants
E144R
1 2 3 4 5 6
Effector constructs coexpressed with the Ubi/LUC reporter
Figure 2-17. Effects of TBP and C-terminal stirrup mutants on
ubiquitin promoter activity in maize cells. Different effectors with
the same amount of DNA were coexpressed with the Ubi/LUC
reporter. Ubiquitin promoter activity in maize cells was expressed
as counts per minute (cpm) of photon emission. The results of one
representative experiment with three replicates are shown.


65
(compare column 3 vs. 2, Figs. 2-7 to 2-11) with ftzQ being the highest (compare
column 3 vs. 2, Fig. 2-7). Although the enhancement in activity is unexplained, a
minimal interpretation is that the E144R mutation of TBP did not impair activated
transcription in vivo.
In contrast to the stimulation in activity obtained with mutant E144R, the E146R
mutation significantly inhibited transcriptional activity for all five activators tested, with
the degree of reduction ranging from more than 2- to about 5-fold (compare column 4
vs. 2, Figs. 2-7 to 2-11). The double mutation El44R/E146R showed a more severe
inhibition ranging from 5- to 16-fold (compare column 5 vs. 2, Figs. 2-7 to 2-11).
Although the E144R mutation alone appeared to stimulate activity, when combined with
E146R (E146R/E144R) transcriptional activity was further reduced from that obtained
from E146R alone. A third site of charge-charge interaction between TBP and TFIIB
involves K197 near the C-terminus of TBP (Nikolov et al., 1995). However, the triple
mutation, E144R/E146R/K197E, showed no further inhibition of transcription compared
to E144R/E146R (compare column 6 vs. 5, Figs. 2-7 to 2-11), suggesting that double
mutation of the stirrup of TBP is sufficient to abolish the TBP-TFIIB interaction in vivo.
Taken together, these results strongly contrast those observed in basal transcription, and
indicate that activated transcription driven by the synthetic GAL4 promoter is highly
dependent on the TBP-TFIIB interaction in vivo.
The inhibition caused by coexpression of TBP with VP 1, VP 16, and ftzQ may be
due to transcriptional squelching defined in an early study (Gill and Ptashne, 1988). In
the TBP titration experiment depicted in Fig. 2-12, ftzQ activity was first enhanced with
low amounts of TBP expression vector and then suppressed when levels of the TBP


33
function of the GAL4 protein is down-regulated by the GAL80 protein in uninduced
cells (inducible by galactose), which binds to the 30 aa at the extreme C-terminal region
of the GAL4 AD (Ma and Ptashne, 1987a). This inhibition can be reversed by
phosphorylation of the GAL4 protein (Parthun and Jaehning, 1992).
The GAL4 DBD consists of two functional motifs: the cysteine-rich region (aa
10-35) and two dimerization domains (aa 50-65 and aa 65-94). In the DBD dimer, each
of the two zinc-fmger-like domains interacts with the major groove of the consensus
GAL4 site (5-CGGAGGACTGTCCTCCG-3) at the CGG triplet of either end
(Marmorstein et al., 1992). A peptide containing the DBD and a single dimerization
domain (aa 50-65) is able to bind DNA, but this interaction is further enhanced by
inclusion of the second dimerization domain (aa 65-94). Because of the high binding
specificity between DNA and the GAL4 DBD, and the lack of recognition of the GAL4
binding site outside of yeast, the GAL4 transcription system has been widely used in
many heterologous organisms to characterize transcriptional activity of many activation
domains. This approach has also been very successful in plant transient expression
systems in characterizing the potential activation domains of plant activator proteins
(Bobb et al., 1995; Nakayama et al., 1997; Schwechheimer et al., 1998; Ulmasov et al.,
1995; Urao et al., 1996).
The 41 aa AD at the C-terminal end of the GAL4 protein is rich of acidic
residues and is classified as an acidic activation domain. Unlike the VP 16 AD, this
domain is thought to form a (3-sheet structure instead of the amphipathic a-helix
(Leuther et al., 1993). However, the mutagenesis analysis on the GAL4 AD showed that
the acidic amino acids present in the motif were not important for activity (Leuther et


135
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Muslin, A. J., Tanner. J. W., Allen, P. M and Shaw, A. S. (1996). Interaction of 14-3-3
with signaling proteins is mediated by the recognition of phosphoserine. Cell 84,
889-897.
Nakayama, T., Okanami, M., Meshi, T., and Iwabuchi, M. (1997). Dissection of the
wheat transcription factor HBP-la(17) reveals a modular structure for the
activation domain. Mol. Gen. Genet. 253, 553-561.
Nakshatri, H., Nakshatri. P., and Currie. R. A. (1995). Interaction of Oct-1 with TFIIB.
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Natesan, S., Rivera, V. M., Molinari, E., and Gilman, M. (1997). Transcriptional
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Nerlov, C., and Ziff, E. B. (1995). CCAAT/enhancer binding protein-a amino acid
motifs with dual TBP and TFIIB binding ability co-operate to activate
transcription in both yeast and mammalian cells. EMBO J. 14, 4318-4328.
Nikolov, D. B., and Burley, S. K. (1994). 2.1 A resolution refined structure of a TATA
box-binding protein (TBP). Nat. Struct. Biol. 1, 621-637.
Nikolov, D. B., Chen, H., Halay, E. D., Usheva, A. A., Hisatake, K., Lee, D. K., Roeder.
R. G., and Burley, S. K. (1995). Crystal structure of a TFIIB-TBP-TATA
element ternary complex. Nature 377, 119-128.
Odell, J. T., Nagy, F., and Chua, N. H. (1985). Identification of DNA sequences
required for activity of the cauliflower mosaic virus 35S promoter. Nature 313,
810-812.
OReilly, D., Hanscombe, O., and OHare, P. (1997). A single serine residue at position
375 of VP16 is critical for complex assembly with Oct-1 and HCF and is a target
of phosphorylation by casein kinase II. EMBO J. 16, 2420-2430.
Ossipow, V., Tassan, J. P., Nigg, E. A., and Schibler, U. (1995). A mammalian RNA
polymerase II holoenzyme containing all components required for promoter-
specific transcription intiation. Cell 83, 137-146.
Parada, C. A., and Roeder, R. G. (1996). Enhanced processivity of RNA polymerase II
triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature
384, 375-378.
Parthun. M. R., and Jaehning, J. A. (1992). A transcriptionally active form of GAL4 is
phosphorylated and associated with GAL80. Mol. Cell. Biol. 12, 4981-4987.


57
indicated that background levels of GUS activity were very low in the Black Mexican
Sweet cell line (column 1, Fig. 2-6). Using the empty vector as the effector, GUS
activity with the CaMV 35S minimal promoter was 12-fold higher than the
promoterless/GUS background (compare column 2 vs. 1, Fig. 2-6). This activity
represents basal transcription in maize cells, since the minimal promoter lacked
upstream elements.
Coexpression of the wild-type AtTBP2 (effector) with the minimal
promoter/GUS reporter stimulated GUS activity 4-fold above levels obtained with the
reporter alone (compare column 4 vs. 2, Fig. 2-6). This result not only indicates that
arabidopsis TBP is compatible with the maize transcription machinery, but that the
elevation in TBP concentration was able to partially compensate for the lack of upstream
elements in the promoter. However, coexpression of AtTFIIB showed no effect on the
transcription (compare column 3 vs. 2, Fig. 2-6), suggesting that TFIIB is not rate
limiting for basal transcription in maize cells.
Next, the functional significance of the TBP-TFIIB interaction was examined in
basal transcription. If the TBP-TFIIB interaction is critical to basal transcription in vivo,
coexpression of the stirrup mutants of TBP with the minimal promoter/GUS reporter
should severely inhibit the basal transcription by competing for TATA binding with the
endogenous TBP. However, over-expression of mutant AtTBP2 with either single
(E144R, E146R), double (E144R/E146R), or triple (E144R/E146R/K197E) point
mutations enhanced basal transcription almost as efficiently as the wild-type TBP
(compare columns 5, 6, 7, and 8 vs. 4, Fig. 2-6). This lack of suppression by stirrup
mutations in TBP suggests that under these conditions of TBP over-expression,



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GUS/LUC
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Transcription activated by Gal4 DBD/VP16
1500-i
1250 -
1000-
750-
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vector
TBP mutants
TBP2
E144R
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Effector constructs coexpressed with GaI4 DBD/VP16
Figure 2-8. Effects of coexpression of TBP or the C-terminal stirrup mutants
on VP 16 transcriptional activity. The details are the same as in Fig. 2-7.


37
protein interactions with other DNA binding factors such as EmBPl (Hill et al., 1996).
The targets for the VP1 activation domain in the PIC are unknown. However, in vitro
binding experiments indicate that it interacts with TFIIB (Baldwin, 1997).
LpHSF8
LpHSF8 is one of the three heat shock transcription factors known in tomato
(Scharf et al., 1990). It is believed that the HSF proteins pre-exist in the cytosol, and are
localized to the nucleus upon heat stress (Scharf et al., 1998). DNA binding activity is
located in the N-terminal portion of the proteins, and a domain responsible for
trimerization (trimerization domain) is adjacent to the DBD at its C-terminal end. HSF
proteins are able to bind to the heat shock-response element of the heat shock genes,
such as hsp70, only in the trimerized state (Kroeger et al., 1993). The tomato HSF8 also
contains an activation domain of aa 394-527 at its C-terminal region rich in acidic
residues interspersed by bulky hydrophobic residues (Treuter et al., 1993). Although
Drosophila HSF has been shown to interact with TBP in vitro (Mason and Lis, 1997),
no information regarding targets with respect to plant HSFs has been reported.
The 14-3-3 Proteins
The 14-3-3s are highly conserved eukaryotic proteins with multiple functions. In
addition to their participation in different signal transduction processes (Braselmann and
McCormick, 1995; Dellambra et al., 1995; Freed et al., 1994; Gelperin et al., 1995),
circumstantial evidence suggest that 14-3-3 proteins may also be involved in
transcriptional regulation in both plant and human systems. Although the 14-3-3
proteins alone do not bind to DNA, several studies have demonstrated that at least one
plant 14-3-3 protein is associated with the G-box DNA binding activity of nuclear


22
Further analysis of the function of human-yeast hybrid TFIIB proteins has revealed a
specie-specific domain for yTFIIB (Shaw et al., 1996). This domain is part of the B1
helix of the first repeat. In addition to possible species specificity, TFIIBs also have
shown promoter-specific responses (Wampler and Kadonaga, 1992). For instance, with
the Drosophila Kr promoter, hTFIIB is unable to substitute in vitro for dTFIIB.
Furthermore, dTFIIB enhances Kr, but suppresses AdE4 promoter activity in a dose-
dependent manner in the same transcription reaction. In contrast, the concentration of
dTFIIB has little effect on activated transcription driven by both VP 16 and Spl
(Wampler and Kadonaga, 1992). The underlying mechanistic differences responsible for
these promoter-specific activities are still unknown.
As shown by many electrophoretic EMSA experiments, TFIIB is able to join the
TBP-DNA complex, but alone does not appear to have affinity for the DNA of the core
promoter. However, a recent study challenges this conclusion by showing that hTFIIB
has specific DNA binding activity (Lagrange et al., 1998). A binding-site selection
assay, which was previously applied in defining the consensus TATA sequence for TBP
binding (Wong and Bateman, 1994), was used to screen for any possible binding
preferences of TFIIB out of 1.7xl07 DNA fragments (Lagrange et al., 1998). After two
rounds of selection, the 5-G/C-G/C-G/A-C-G-C-C-3 sequence appeared to specifically
associate with TFIIB in terms of statistically significant frequency, and is defined as the
IIB recognition element (BRE) (Lagrange et al., 1998). The BRE is located at -32 to -38
base pair upstream of the TATA motif of the adenovirus major late promoter. The
guanine at -34 (5-G/C-G/C-G/A-C-G-C-C-3) is thought to be the most critical base of
the BRE (Lagrange et al., 1998). The specificity of TFIIB-BRE binding was confirmed


2
on the core promoter can be facilitated by a transcriptional activator or inhibited by a
transcriptional repressor, which results in transcriptional activation or repression
respectively.
Initial biochemical characterization studies of several in vitro transcription
systems suggest that many general transcription factors together with RNA polymerase
II assemble on the promoter of a DNA template in a stepwise fashion in order for
transcription to occur (Weinmann, 1992). These experiments were primarily based on
the chromatographic purification of nuclear proteins. Using various ionic exchange
columns, RNA polymerase II and several general transcription factors were purified
from the nuclear extracts. Their designations were initially determined by their order of
elution and are as follows: TFIID (Matsui et al., 1980), TFIIA (Cortes et al., 1990;
Samuels and Sharp, 1986), TFIIB (Ha et al., 1991; Matsui et al., 1980), TFIIE (Flores et
al., 1989; Inostroza et al., 1991), TFIIF (Flores et al., 1990) and TFIIH (Flores et al.,
1992). By sequential addition of the purified enzyme and factor proteins, in vitro PIC
assembly can be accessed. However, the high order PIC complex is formed only when
these proteins are added into the reaction in the fixed orders (Flores et al., 1992). These
observations led to the hypothesis that formation of a functional PIC involves the
multistep addition of general transcription factors in an ordered sequence.
According to the multistep hypothesis based on in vitro transcription systems in
both HeLa and yeast, the TATA binding protein (TBP) of TFIID is the first protein to
recognize the DNA TATA motif of the core promoter (Buratowski et al., 1989;
Horikoshi et al., 1989; Maldonado et al., 1990). Following TBP binding to TATA,
TFIIA and TFIIB interact with both TBP and DNA to form DAB complex (Buratowski


105
with human TAFh32 and TAFh55 synthesized by a coupled transcription/translation
system. Human 14-3-3 u interacted with human TBP (compare lane 4 vs. 3, Fig 3-5A),
TFIIB (compare lane 2 vs. 1, Fig 3-5A) and positive co-factor hTAF32 (compare lane 1
vs. 2, Fig 3-5B), but not with hTAFn55 (lane 3, Fig 3-5B). This multiple interaction
pattern of hi 4-3-3 u with the human PIC components parallels that previously observed
for VP16 (Chiang and Roeder, 1995; Kim and Roeder, 1994; Klemm et al., 1995;
Roberts et al., 1993).
Human 14-3-3 u Contains Two Domains That Bind TFIIB
Using human proteins, regions of 14-3-3 u required for binding TFIIB were
identified. A series of deletion mutants for the 14-3-3 d were constructed as GST
fusions such that the truncations did not intrude into regions with a-helical structure
(each 14-3-3 monomer includes 9 a-helices). TFIIB showed affinity for both the N- and
C-terminal halves of the immobilized 14-3-3 protein (lanes 6 and 7, Fig. 3-6). Further
progressive deletion for the C-terminal half of 14-3-3 (helices 5 through 9) identified a
single binding domain for TFIIB corresponding to helix-7, with helices 5, 6, 8, and 9
being dispensable (compare lanes 4, 5, 11, 12, 13, and 16 vs. 15, Fig. 3-6). Progressive
deletion of the N-terminal helices (helices 1 through 4) indicated that a second TFIIB
binding domain is located within helices 2 and 3 (compare lane 10 vs. 8 and 9, Fig. 3-6).
Helices 2 and 3 appear to comprise a single functional unit required for TFIIB binding,
since constructs containing only one helix were unable to bind (lanes 2 and 3, Fig. 3-6).
Alanine Substitutions in 14-3-3 Helix 7 Identify Amino Acids Critical for Binding TBP
and TFIIB
A detailed mutagenesis of 14-3-3 helix 7 was performed to identify aa residues
required for binding TBP and TFIIB. These studies were conducted using plant proteins:


GUS/LUC
64
Transcription activated by Gal4 DBD/VP1
Figure 2-11. Effects of coexpression of TBP or the C-terminal stirrup
mutants on VP1 transcriptional activity. The details are the same as in
Fig. 2-7.


96
(Gal4 DBD or Gal4 DBD/14-3-3). The bombarded onion tissues were allowed to
recover for 40 hr in the dark at 25C before harvesting.
The GUS and luciferase activities were analyzed by the same assays described
in chapter 2 of this dissertation.
Results
Plant 14-3-3 Proteins Interact with Human TFIIB in vitro
To test if plant 14-3-3 proteins may directly participate in transcriptional
activation by the promoter complex which they are associated with, we employed the in
vitro protein-protein interaction approach using GST pull-down assays to examine
possible interactions between 14-3-3 proteins and components of the PIC.
In the first in vitro binding experiment, interaction between human TFIIB and
maize 14-3-3 protein was tested. hTFIIB was expressed in E. coli as a GST-fiision and
immobilized on glutathione beads. Maize 14-3-3 (His-tagged) was expressed in E. coli
and purified by Ni2+-beads. Possible interaction between hTFIIB and maize 14-3-3 was
examined by incubating the free maize 14-3-3 protein with bead-immobilized GST-
hTFIIB or GST control proteins. After extensive washing of unbound molecules, the
bound 14-3-3 was detected by immunoblots with anti-14-3-3 polyclonal antibody. As
shown in Fig. 3-1 A, maize 14-3-3 was retained on GST-hTFIIB beads, but not on the
GST control beads. This result was an indication that the maize 14-3-3 protein
physically interacted with hTFIIB, but not with GST. Further experiments showed that
this interaction was stable under high salt concentration up to 0.5 M, although binding
was gradually reduced with increasing salt concentrations (data not shown).


133
Lu. G., DeLisle. A. J.. de Vetten, N. C., and Ferl, R. J. (1992). Brain proteins in plants:
an Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA
binding complex. Proc. Natl. Acad. Sci. USA 89, 11490-11494.
Luo, Z., Zhang, X., Rapp. U., and Avruch, J. (1995). Identification of the 14-3-3
domains important for self-association and Raf binding. J. Biol. Chem. 270,
23681-23687.
Lyons, J. G., and Chambn, P. (1995). Direct activation and anti-repression functions of
GAL4-VP16 use distinct molecular mechanisms. Biochem. J. 312, 899-905.
Ma, J., and Ptashne, M. (1987a). The carboxy-terminal 30 amino acids of GAL4 are
recognized by GAL80. Cell 50, 137-142.
Ma. J., and Ptashne. M. (1987b). Deletion analysis of GAL4 defines two transcriptional
activating segments. Cell 48. 847-853.
Maldonado, E., Ha, L, Cortes, P., Weis, L., and Reinberg, D. (1990). Factors involved in
specific transcription by mammalian RNA polymerase II: role of transcription
factors IIA, IID, and IIB during formation of a transcription-competent complex.
Mol. Cell. Biol. 10. 6335-6347.
Malik, S., Lee, D. K., and Roeder, R. G. (1993). Potential RNA polymerase II-induced
interactions of transcription factor TFIIB. Mol. Cell. Biol. 13, 6253-6259.
Marmorstein, R., Carey, M., Ptashne, M and Harrison, S. C. (1992). DNA recognition
by GAL4: structure of a protein-DNA complex. Nature 356, 408-414.
Marra, M., Fullone, M. R., Fogliano, V., Pen, J., Mattei, M., Masi, S., and Aducci, P.
(1994). The 30-kilodalton protein present in purified fusicoccin receptor
preparations isa 14-3-3-like protein. Plant Physiol. 106, 1497-1501.
Marrs, K. A., Casey, E. S., Capitant, S. A., Bouchard, R. A., Dietrich, P. S., Mettler. I.
J., and Sinibaldi, R. M. (1993). Characterization of two maize HSP90 heat shock
protein genes: expression during heat shock, embryogenesis, and pollen
development. Dev. Genet. 14, 27-41.
Martinez, E., Chiang, C. M., Ge, H., and Roeder, R. G. (1994). TATA-binding protein-
associated factor(s) in TFIID function through the initiator to direct basal
transcription from a TATA-less class II promoter. EMBO J. 13, 3115-3126.
Mason, P. B., Jr., and Lis, J. T. (1997). Cooperative and competitive protein interactions
at the hsp70 promoter. J. Biol. Chem. 272, 33227-33233.


82
limiting for basal transcription in vivo. This implies that recognition of the TATA motif
by TBP or TFIID may be the most important step determining levels of basal
transcription.
In contrast to the in vitro system, basal transcription in maize cells appears not to
require the TBP-TFIIB interaction. This conclusion is evident since AtTBP2 mutations
at the C-terminal stirrup did not interfere with the positive function of TBP in basal
transcription. Stimulation of this in vivo basal activity by exogenous TBP was
comparable between the wild type and the mutant proteins as shown in Fig. 2-6.
However, reconstituted in vitro transcription using human components indicates that a
single point mutation in the C-terminal stirrup of hTBP is sufficient to abolish basal
transcription (Bryant et al, 1996). These opposing results indicate that fundamental
differences may exist in the properties of basal transcription between plants and humans
or, alternatively, between in vivo and in vitro transcription systems. Regardless of the
nature of differences with other organisms, it is apparent from these results that an
alternative interaction pathway is available in plant cells for the PIC assembly to support
basal transcription when the TBP-TFIIB interaction is disrupted.
The TBP-TFIIB interaction is critical for function when a synthetic GAL4
promoter is activated by a single type of activation domain. In this simple experimental
system, either holoenzyme or TFIID is initially recruited to the promoter. Once this
occurs, the rate-limiting step is likely in the subsequent recruitment of the second
complex (Gonzalez-Couto et al, 1997). Under these conditions, the strength of
interaction between TFIID and holoenzyme (TFIIB) may then be correlated with the
level of transcriptional activity. Although many interactions among general transcription


101
mutant of hTFIIB were tagged with the T7 epitope at their C-terminal ends and were
added as crude E. coli extracts to binding reactions containing immobilized GST-VP16
or GST-14-3-3. The VP16 mutant VP16A456/F442P has the C-terminal activation motif
deleted and the critical residue, phenylalanine 442, of the N-terminal activation motif is
substituted by proline. This VP 16 mutant was shown to lose its transactivation potential
and ability to interact with hTFIIB as well (Lin et al., 1991). Therefore, GST-
VP16A456/F442P was used as the negative control for hTFIIB binding in this
experiment. As shown in Fig. 3-3, both wild type and R185E/R193E mutant of hTFIIB
did not bind to GST-VP16A456/F442P, but did bind to both GST-VP16 and GST-14-3-
3. The affinities of hTFIIB for wild type VP 16 and 14-3-3 were approximately the same.
The R185E/R193E mutation of hTFIIB impaired interactions with VP16 and 14-3-3 to a
similar degree. These results suggest that both VP 16 and 14-3-3 may use the same or
similar mechanisms in binding to hTFIIB, and require R185, or R193, or both as
important residues within hTFIIB for interactions. Additionally, the binding of hTFIIB
to GST-14-3-3 in this experiment is reciprocal to that of 14-3-3 to GST-hTFIIB as
shown in Fig. 3-2, suggesting that the GST moiety was not involved in the interaction
between hTFIIB and arabidopsis 14-3-3 .
Since amino acid residues R185 and R193 of hTFIIB were implicated in the
binding of both VP 16 and arabidopsis 14-3-3 <|>, VP16/hTFIIB and 14-3-3/hTFIIB
interactions may be mutually exclusive. To test this idea, the binding competition
experiments were conducted as shown in Fig. 3-4. In the presence of an excess amount
of free 14-3-3, binding of hTFIIB to immobilized GST-VP16 was greatly inhibited
(compare lane 3 vs. 2, Fig. 3-4A). Hypothetically, the observed inhibition could be due


69
Gal4 DBD/ftzQ activity in TGTA/TBPm3
coupled transcription system
o
z>
_J
CO
3
o
70
60
50
40
30
20
10
0
T7
vector
1 2 3 4 5 6 7
TATA
reporter
TGTA reporter
Figure 2-13. Effects of TBPm3 and its C-terminal stirrup mutants on ftzQ
transcriptional activity in the TGTA/TBPm3 coupled system in maize cells.
AtTBP2 was mutated by three amino acid substitutions within its concave
DNA binding surface to generate TBPm3 (Strubin and Struhl, 1992). In the
context of this TBPm3, single, double, or triple point-mutation was
introduced into the C-terminal stirrup as labeled in the figure. The same
amounts of different effector plasmids were coexpressed with the reporter
containing the mutated TATA motif, TGTA. Column 1 was the control for
normal ftzQ activity using the wild type TATA reporter. ftzQ activity was
expressed as relative GUS/LUC activity. The results of one representative
experiment with three replicates are shown. The data for each treatment was
the average of three replicates.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FUNCTIONAL ANALYSIS OF ARABIDOPSIS GENERAL
TRANSCRIPTION FACTORS TBP AND TFIIB
By
Songqin Pan
May 1999
Chairman: William B. Gurley, Ph.D.
Major Department: Plant Molecular and Cellular Biology Program
Studies with human, Drosophila and yeast systems have demonstrated that the
general transcriptional factors, TBP and TFIIB, play crucial roles in the transcription of
class II genes in eukaryotes. These two proteins not only are important in assembly of
the pre-initiation complex (PIC), but also serve as critical targets for recruitment by
activation domains in activated transcription. To evaluate the role of TBP-TFIIB
interaction in transcription, a series of mutations in the C-terminal stirrup of arabidopsis
TBP2 designed to disrupt TBP-TFIIB interaction were introduced into plant cells
(maize) using transient assays. TBP-TFIIB interaction was shown to be dispensable for
basal transcription, but required for activated transcription driven by different types of
activation domains in the GAL4-dependent transcription system. Inhibition of
transcription by TBP mutation ranged from 5- to 16-fold indicating that association of
TBP and TFIIB varied in importance depending on the activation domains. In vitro
protein binding experiments showed that both El46 and El44 of AtTBP2 are essential
IX


TFIIIB TBP-containing factor for RNA polymerase III
TGTA mutated TATA
TRF TBP-related factor
pg microgram
pi micro liter
VP1 viviparous protein 1
VP 16 herpes simplex virus protein 16
viii


89
1995; Toker et ai, 1992). In animal brain cells, 14-3-3 proteins can activate
phosphorylated tyrosine tryptophan hydroxylase (TPHase), which is an important
enzyme involved in neural transmission (Ichimura et ai, 1987; Yamauchi et ai, 1981).
In permeabilized adrenal chromafm cells, 14-3-3 proteins are able to mediate calcium-
dependent exocytosis (Morgan and Burgoyne, 1992). In yeast, 14-3-3s are required for
cell viability (Van Heusden et ai, 1994) and play a role in a mechanism that acts as
checkpoint for mitotic DNA damage repair (Ford et ai, 1994). 14-3-3 proteins also
appear to have chaperone-like function since they can facilitate protein translocation
through the mitochondrial membrane by their ATPase activity (Alam et ai, 1994). In
plants, 14-3-3 proteins are the receptor for the phytotoxin fusiccoccin (Marra et ai,
1994), and one iso form specifically inhibits nitrate reductase activity from spinach cells
(Bachmann et ai, 1996). In several species which include dicots and monocots, 14-3-3
proteins are found in a sequence-specific DNA binding complex in vitro (Lu et ai,
1992; de Vetten et ai, 1992) and are localized in the nucleus in vivo (Bihn et ai, 1997).
Although the 14-3-3 protein family appears to be involved in a wide range of biological
functions, a common theme regarding function is their property for interactions with
other proteins (Ferl, 1996).
Biochemical characterizations of the 14-3-3 proteins have shown that they are
able to form homo- or heterodimers through their N-terminal dimerization domains
(Jones et ai, 1995; Luo et ai, 1995; Wu et ai, 1997a). The 14-3-3 protein dimer is cup
shaped containing a spacious internal area and negative residues located in the C-
terminal portion of each monomer. These charged residues align on the surface to form a
negative groove (Liu et ai, 1995). This topological feature is predicted to be highly


97
A. Immobilized GST-hTFIIB or GST; 14-3-3 free
1% input GST GST-hTFIIB
1 2
maize 14-3-3
B. Immobilized GST-hTFIIB or GST; 14-3-3 free
Atl 4-3-3
V x <|> ffl V) Atl4-3-3
isoforms
Figure 3-1. In vitro interactions of human TFIIB with 14-3-3s from maize and
arabidopsis. The hTFIIB coding sequence was expressed in E. coli as a GST
fusion. GST-hTFIIB or GST was immobilized on glutathione-agarose beads and
incubated with different isoforms of arabidopsis and maize 14-3-3 proteins
purified from E. coli. Binding reactions were conducted at room temperature for
3 hr in buffer containing 0.10 M KC1 and 0.5% BSA. 14-3-3 isoforms were
incubated with equal amounts (5 fig) of GST or GST-hTFIIB during the binding
reaction. After extensive washing, bound 14-3-3 proteins were eluted and
detected by western blotting using anti-14-3-3 polyclonal antibody. (A) Maize
14-3-3 protein interacted with GST-hTFIIB (lane 2) but not with the GST control
(lane 1). (B) Five arabidopsis 14-3-3 isoform proteins interacted with GST-
hTFIIB, but not with GST alone (control lanes; c).


55
about 50% efficiency (Fig. 2-4B). Similarly, a human TFIIB deletion mutant showed a
40% efficiency in binding with TBP when its C-terminal 30 or 80 residues were
truncated (Ha et al., 1993).
In a reciprocal experiment (Fig. 2-5) where GST-TFIIB was immobilized on
beads and the TBP is free in solution, similar results were obtained: C-terminal
mutations of TBP interfered TBP/TFIIB interactions. Both wild type and mutants of
TBP did not bind to GST control (data not shown), but bound to GST-TFIIB (Fig. 2-5).
Binding of TBP to GST-TFIIB was inhibited by TBP C-terminal stirrup mutations.
Inhibition by TBP mutant E146R remained at about 50%; however, inhibition by the
E144R mutation was more severe when TFIIB was immobilized, with a 7-fold reduction
seen in binding. Inhibition by the double TBP mutation (E144R/E146R) in this
experiment was more than 8-fold, which was almost equal to that obtained in the
reciprocal configuration shown in Fig. 2-4. Although mutation of the two glutamic acid
residues in the C-terminal stirrup of arabidopsis TBP2 did not completely abolish
interactions with TFIIB, it is clear that mutation of these residues is severely disruptive
to binding, as is the case with human and yeast proteins (Lee and Struhl, 1997; Tang et
al., 1996).
The TBP-TFIIB Interaction is Dispensable in Basal Transcription in vivo
Basal transcription in maize suspension cells was determined using the GUS
reporter gene under control of the CaMV 35S minimal promoter (nt -46) which contains
no activation elements upstream of the TATAA motif (minimal promoter) (Odell et al.,
1985). The same reporter with the promoter deleted served as the negative control for no
transcription (promoterless/GUS). Experiments with the promoterless/GUS reporter


51
GUS and Luciferase Assays
The luciferase assay was conducted using a multipurpose scintillation counter
(Beckman) to measure single photon emission from the enzymatic reaction in which 5
pi of maize cell extract was incubated with 25 pi of luciferase substrate (Promega) at
room temperature. Light emission was measured immediately after the mixing of extract
and substrate.
To measure GUS activity, 50 pi of the original extract was incubated with 75 pi
of 2 mM 4-Methylumbelliferyl P-D-Glucuronide (MUG) (GUS substrate) at 37C for
2.25 hours, and the reaction was stopped by mixing 50 pi of the extract/substrate
mixture with 950 pi of 0.2 N Na2C03. The product of the enzymatic reaction 4-
Methylumbelliferone (MU) was measured using a spectrofluorophotometer (Shimadzu,
RF5000) with excitation wavelength at 365 nm and emission wavelength at 445 nm
respectively. The MU concentration for the first 15 min of the reaction was taken as the
background and subtracted from the final Mu concentration after the 2.25 hr reaction.
Protein Expression and Purification from E. coli
GST-AtTFIIB and GST-AtTBP2 or its mutants were expressed in E. coli using
the pGEX-2TK expression vector (Pharmacia). The overnight grown bacterial cells were
diluted 1:100 in 50 ml Luria Broth (LB) medium containing 100 pg/ml ampicilin, and
then allowed to grow for 3 hr at 37C. Protein expression was induced with 10 pM
isopropyl-1-thio-P-D-galactoside (IPTG) for 7 hr at room temperature. All IPTG-
induced E. coli cells were collected by centrifugation, washed with cold lx PBS buffer,
and suspended in 1 ml of cold lx protein binding buffer. The lx protein binding buffer
contained 20 mM (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) (HEPES),


123
Buratowski, S., and Zhou. H. (1993). Functional domains of transcription factor TFIIB.
Proc. Natl. Acad. Sci. USA 90, 5633-5637.
Burley, S. K.. and Roeder, R. G. (1996). Biochemistry and structural biology of
transcription factor (ID (TF1ID). Annu. Rev. Biochem. 65. 769-799.
Bums L. G.. and Peterson, C. L. (1997). The yeast SWI-SNF complex facilitates binding
of a transcriptional activator to nucleosomal sites in vivo. Mol. Cell. Biol. 17,
4811-4819.
Cadena. D. L., and Dahmus, M. E. (1987). Messenger RNA synthesis in mammalian
cells is catalyzed by the phosphory lated form of RNA polymerase 11. J. Biol.
Chem. 262, 12468-12474
Carey, M., Kakidani. H.. Leatherwood, J., Mostashari. F., and Ptashne. M. (1989). An
amino-terminal fragment of GAL4 binds DNA as a dimer. J. Mol. Biol. 209,
423-432.
Chao, D. M.. Gadbois. E. L., Murray, P. J.. Anderson. S. F., Sonu, M. S., Parvin, J. D.,
and Young, R. A. (1996). A mammalian SRB protein associated with an RNA
polymerase II holoenzyme. Nature 380, 82-85.
Chatteijee, S.. and Struhl, K. (1995). Connecting a pro mo ter-bound protein to TBP
bypasses the need for a transcriptional activation domain. Nature 374, 820-822.
Chen, J. L., Attardi, L. D., Verrijzer, C. P., Yokomori, K.., and Tjian, R. (1994).
Assembly of recombinant TFIID reveals differential coactivator requirements for
distinct transcriptional activators. Cell 79, 93-105.
Chen, X., Farmer, G., Zhu, H., Prywes, R., and Prives, C. (1993). Cooperative DNA
binding of p53 with TFIID (TBP): a possible mechanism for transcriptional
activation. Genes Dev. 7, 1837-1849.
Chiang, C. M., and Roeder, R. G. (1995). Cloning of an intrinsic human TFIID subunit
that interacts with multiple transcriptional activators. Science 267, 531-536.
Chicca, J. J. 2nd, Auble, D. T., and Pugh. B. F. (1998). Cloning and biochemical
characterization of TAF-172, a human homo log of yeast Motl. Mol. Cell. Biol.
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IIF. J. Biol. Chem. 272, 11495-11502.


93
transformed into E. coli strain BL21(DE3). Transformants grown overnight were diluted
1:100 in 50 ml of LB containing appropriate antibiotics, and grown to an optical density
of 0.6 (OD600-O.6) before IPTG induction. The expression of His- or T7-tagged fusion
proteins was induced by 0.4 mM IPTG for 3 hr at 37C. All IPTG-induced E. coli cells
were collected by centrifugation, washed with cold lx PBS buffer, and suspended in 1
ml of cold lx protein binding buffer (described below). The cells were disrupted by four
one minute bursts of a probe sonicator, then centrifuged for 30 min at 12,000 rpm.
Soluble proteins in the supernatants were either used directly in protein binding assays
or were purified by their specific affinity tags.
Protein Purification from E. coli Lysate
GST fusion proteins from E. coli lysates were purified by incubation with
glutathione-agarose beads (Pharmacia) at 4C for 30 min with continuous rotation. The
beads were pelleted by brief microcentrifugation, then washed with cold lx protein
binding buffer plus 0.5 M KC1 for 10 min, followed by a final wash with cold lx protein
binding buffer. The beads containing the purified GST fusion proteins were suspended
in the lx protein binding buffer plus 20% glycerol and stored at -20C for use. When
necessary, GST-fiision proteins were eluted from beads by 0.2 M free glutathione at 4C
for 30 min, and then dialyzed against 0.5 liter of the lx protein binding buffer for
overnight with three changes. In some cases, recombinant proteins were cleaved from
the GST moiety by thrombin digestion in the lx protein binding buffer at 4C overnight.
His-tagged proteins were purified by, and eluted from Ni2+-beads according to the
manufacturer recommendations (Novagen). T7-tagged proteins were purified by protein
A conjugated beads coated with T7 antibody, which served as a bridge between protein


38
extracts from arabidopsis (Lu et al., 1992), maize (de Vetten and Ferl, 1994) and rice
(Schultz et al., 1998). Consistent with a role in regulating gene expression, 14-3-3
iso forms are found in the nuclei of both arabidopsis and maize cells in vivo (Bihn et al.,
1997). More recently, different 14-3-3 iso forms were found to directly interact with two
G-box-related transcription factors, VP1 and EmBPl, with different affinities (Schultz et
al., 1998). In animal systems, human 14-3-3 r\ is found to interact with another
transcription factor, glucocorticoid receptor, and stimulate glucocorticoid receptor-
dependent transcription in COS-7 cells (Wakui et al., 1997). It is possible that 14-3-3
proteins may affect transcription as part of a DNA-protein complex that may associate
with other transcriptional regulatory proteins to either regulate the activity of the
complex, or directly contact the PIC components and influence the PIC assembly.
Rationale for the Present Study
Transcriptional regulation has been an extensive subject of both human and yeast
research for more than a decade. However in plants, related research has mostly limited
to the characterization of promoter elements. There has been little information available
to help design a high-output-expression system or provide specific control of gene
expression at the level of manipulating the function and activity of the transcription
machinery. The results from studies in human and yeast systems sometimes appear to be
controversial, suggesting that different kingdoms may have unique aspects of
transcriptional regulation in addition to the common requirements. These considerations
resulted in a strong encouragement to initiate this project to study the functional roles of
plant TBP and TFIIB proteins, which appear to be the most effective factors for control
of PIC function. The following chapters will describe the findings of this study.


4
promoter by TFIID and the association of TFIID with the holoenzyme mediated by the
interactions between their respective components.
From the finding of the holoenzyme, one would expect that the recruitment of
any components of the two megacomplexes, TFIID and holoenzyme, to the promoter to
correctly assemble the PIC should be able to activate transcription. In contrast, the
stepwise model predicts that only the recruitment of TFIID, which is involved in the
very first step of the assembly pathway of the PIC, would most significantly stimulate
transcription. This hypothesis has been tested by several labs with the approach of
artificial recruitment in which a component of either TFIID or holoenzyme is directly
fused to a DNA binding domain (DBD). The tested components include TBP (Chatterjee
and Struhl, 1995; Klages and Strubin, 1995; Xiao et al., 1997; Xiao et al., 1995b),
TAFns (Gonzalez-Couto et al., 1997), TFIIB (Gonzalez-Couto et al., 1997; Lee and
Struhl, 1997) and mediator proteins (Barberis et al., 1995; Gaudreau et al., 1997). All of
them show strong stimulation of transcription when tethered to a DNA binding element,
and the level of stimulation is comparable to that obtained by a typical activation
domain such as VP 16 (Gonzalez-Couto et al., 1997). These experiments have clarified
that formation of the PIC does not require stepwise recruitments in vivo. Instead,
recruitment of a single component of either the holoenzyme or TFIID can lead to
assembly of a functional pre-initiation complex on the promoter. Recruitment of first
complex, either TFIID or the holoenzyme, presumably results in subsequent recruitment
of the second, which may be mediated by many protein-protein interactions between
their components as characterized in vitro (Goodrich et al., 1993; Ha et al., 1993;
Usheva et al., 1992).


139
Tansey, W. P., and Herr. W. (1995). The ability to associate with activation domains in
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transcription in vivo. Proc. Natl. Acad. Sci. USA 92, 10550-10554.
Tansey, W. P., and Herr, W. (1997). Selective use of TBP and TFIIB revealed by a
TATA-TBP-TFIIB array with altered specificity. Science 275, 829-831.
Thompson, C. M., and Young, R. A. (1995). General requirement for RNA polymerase
II holoenzyme in vivo. Proc. Natl. Acad. Sci. USA 92. 4587-4590.
Timmers, H. T., Meyers, R. E., and Sharp, P. A. (1992). Composition of transcription
factor B-TFIID. Proc. Natl. Acad. Sci. USA 89. 8140-8144.
Toker, A., Sellers, L. A., Amess, B., Patel, Y., Harris, A., and Aitken, A. (1992).
Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep
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Treuter, E., Nover, L.. Ohme, K., and Scharf, K. D. (1993). Promoter specificity and
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Genet. 240, 113-125.
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VP 16. Genes Dev. 2, 730-742.
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transcriptional activation domain of ATMYB2, a drought-inducible Arabidopsis
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Usheva, A., Maldonado, E., Goldring, A., Lu, H., Houbavi, C., Reinberg, D and Aloni,
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53.


115
The demonstration that 14-3-3 proteins have affinity for components of the PIC suggests
that 14-3-3 proteins may function as a bridge between activators and general
transcription factors to facilitate recruitment of the PIC to the promoter. In this regard,
14-3-3 proteins may act as co-activators.
The domains within 14-3-3 for binding activators and general transcription
factors appear to be distinct. For example, the present study has shown that helix-7 of
the box-1 domain is the primary site for binding general transcription factors TBP and
TFIIB; however, the N-terminal region seems to be important in mediating VP1 binding
(Schultz et al., 1998). The existence of more than one 14-3-3 domain for protein
interaction is consistent with their postulated role as coactivators, since multiple protein
contacts may be required for coactivator function as a bridging protein. The
demonstration of 14-3-3 affinity for general transcription factors raises the possibility
that stimulation of glucocorticoid receptor-dependent transcription by hi 4-3-3 r| may be
due to its potential for recruiting general transcription factors instead of due to a
signaling role as suggested (Wakui et al., 1997).
Interactions between 14-3-3s and general transcription factors appear to be a
conserved phenomenon rather than isoform specific. The seven tested 14-3-3 proteins
from arabidopsis, maize and humans all have affinity for TFIIB, consistent with the
highly conserved protein structure of the family. Although human 14-3-3 r| was not
tested in this study, it seems probable that it also has the ability to interact with TBP and
TFIIB, because it shows 100% amino acid identity in helix-7. Additionally, these
interactions are not phosphoserine dependent since TBP, TFIIB and hTAFu32 do not
contain any potential RSXpSXP motifs in their amino acid sequences. Together with


59
TBP/TFIIB interactions only play a minor role, if any, in supporting basal transcription.
The stimulation of basal transcription by over-expression of TBP argues that the
arabidopsis TBP was both expressed and functional for wild type and mutant forms of
the protein.
The TBP-TFIIB Interaction is Required for Activated Transcription by the GAL4
Promoter
To address whether the TBP-TFIIB interaction is important for the activated
transcription, experiments were conducted using the GAL4-driven reporter system
(GAL4xlO/GUS). The activator proteins were heterologous fusions between the Gal4
DBD and several different types of activation domains including the acidic activation
domains VP 16 (mammalian virus) and GAL4 (yeast), the glutamine-rich activation
domain of ftzQ (Drosophila), and two activation domains from plant transcription
activators, VP1 (maize) and LpHSF8 (tomato heat shock factor). In separate
experiments, these activators from either plants or the more widely diverging organisms
were determined to function in the maize Black Mexican Sweet cell line, showing strong
activation of transcription when compared to Gal4 DBD alone (data not shown).
Transcriptional activity of these activation domains was then monitored by coexpression
of TBP, or its C-terminal stirrup mutants, with each activator in five parallel
experiments shown in Figs. 2-7 through 2-11. Coexpression of wild type TBP slightly
inhibited activity of ftzQ (compare column 2 vs. 1, Fig. 2-7), VP 16 (compare column 2
vs. 1, Fig. 2-8) and VP1 (compare column 2 vs. 1, Fig. 2-11) by approximately 25%, but
enhanced Gal4 AD (compare column 2 vs. 1, Fig. 2-9) and LpHSF8 (compare column 2
vs. 1, Fig. 2-10) activities by more than 60%. Compared to wild type TBP, the E144R
mutation showed variable levels of enhancement for all five activation domains


103
bead
O
GST-VP16
O+/-0
T7-TFIIB 14-3-3
GST GST-VP16
B.
i
5%
TFIIB GST-VP16 GST competitor
input (5x) (5x)
a-T7
(jZD=|7ZZZZZZA
igG
protein A T7-14-3-3
M hTFIIB
0+[0o]
TFIIB GST- GST
igG
VP 16
Protein A-
immobilized
14-3-3
Figure 3-4. Competition between 14-3-3 and VP 16 for hTFIIB binding. (A) T7-
tagged hTFIIB was incubated at room temperature for 3 hr with immobilized GST-
VP16 in the absence (lane 2) or presence (lane 3) of a 10-fold excess (compared to
VP 16) of 14-3-3 protein in binding buffer containing 0.15 M KC1. Bound hTFIIB
was detected on western blots using anti-T7 antibody. (B) Human TFIIB was
incubated with 14-3-3 (T7-tagged) immobilized on protein A Sepharose beads in
the presence of GST-VP6 or GST. GST-VP6 and GST were in 5-fold excess to the
immobilized 14-3-3 protein in binding buffer containing 0.15 M KC1 at 4C for 3
hr. The bound hTFIIB molecules were detected using anti-hTFIIB monoclonal
antibody (Promega). Large and small chains of T7-antibody released from the
Sepharose beads were also detected by the secondary antibody in the western blot
as indicated.


70
90
80
O
3
_J
CO
3
CD
40
30
20
10
70
60
50
0
T7
vector
Gal4 DBD/VP1 activity in TGTA/TBPm3
coupled transcription system
TBPm3
wt
E144R
E!46R E146R
reporter
TGTA reporter
Figure 2-14. Effects of TBPm3 and its C-terminal stirrup mutants on
VP1 transcriptional activity in the TGTA/TBPm3 coupled system in
maize cells. The details are the same as in Fig. 2-13.


GUS (nM Mu/hr)
72
B.
400000
350000
300000
250000
200000
150000
100000
50000
0
T7
vector
TBPm3
1 2 3 4 5 6 7
TATA I
reporter TGTA reporter
Figure 2-15 -- continued
(B) The same as in (A) except that GUS activity was not normalized by
luciferase activity of the internal control.


32
Transacting Proteins
A typical transcriptional activator protein contains two functional domains: the
DNA binding domain and the transcriptional activation domain. The two domains often
are separable from each other and can usually reconstitute transcriptional activity when
any of the two types are fused together. In my study using plant cells, the DNA binding
domain from the GAL4 activator protein (Marmorstein et al., 1992) was translationally
fused to the activation domains from several other sources to activate transcription of a
reporter gene containing GAL4 binding sites upstream of the TATA motif. The chimeric
activators used in this study include the well characterized activation domains of VP 16
from human herpes simplex virus, GAL4 from yeast and Ftz from Drosophila as well as
several others from plants with less characterized features including VP1 from maize,
HSF from tomato and a 14-3-3 protein from arabidopsis. The following sections attempt
to summarize the important features for each activator protein (or potential activator
protein) used in this study.
GAL4
The GAL4 protein is the transcriptional activator required for expression of the
galactose/melibiose regulon in yeast in order to regulate carbohydrate metabolism
(Laughon and Gesteland, 1982). The protein consists of 881 amino acids. The N-
terminal 1-65 aa comprises the DNA binding domain (DBD) recognizing the GAL4 site
in the upstream activation sequences of the structural genes of the regulon (Marmorstein
et al., 1992). The C-terminal portion of the protein contains two activation domains
(AD) (Ma and Ptashne, 1987b), and the large middle portion of the protein can be
deleted without affecting transcription (Ma and Ptashne, 1987b). The transcriptional


GUS (nM Mu/hr)
75
B.
160000
TBP mutants
E144R
Effector constructs coexpressed
with the CaMV 35S/GUS reporter
Figure 2-16 continued
(B) The same as in (A) except that GUS activity was not normalized
by luciferase activity of the internal control.


132
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15
The equilibrium between monomer and dimer forms may be important in regulation of
the TBP-DNA interaction, since this interaction is a slow reaction as demonstrated in
kinetic studies (Coleman and Pugh, 1997). As a monomer, the TBP molecule recognizes
the TATA motif (5-TATAAAAG-3) at its minor groove when bound to the AdMLP
core promoter (Kim and Burley, 1994). The protein sits astride the DNA with its first
and second direct repeats interacting with the 3 and 5 halves of the eight base pairs of
DNA, respectively. The protein-DNA interaction is largely mediated by hydrophobic
van der Waals contacts and involves an induced-fit mechanism. It is thought that the 5
half of the TATA element first makes contact with the underside of the second direct
repeat of TBP. As a result of this interaction, both DNA and the protein undergo
significant conformational changes to allow the best fit for the second interaction
between the 3 half of DNA and the first direct repeat of TBP to result in a overall tight
association between the two molecules. The conformational changes in DNA are
characterized as the partially unwound center of the eight base pairs, the widened minor
groove, the severe bend towards the major groove and the severe twist at either end of
the TATA. The insertions of phenylalanines 148 and 165 into the first base pair and
phenylalanines 57 and 74 into the last base step result in two strong kinks in the DNA
that are believed to be critical in distorting the DNA. As a consequence of protein-DNA
interaction, there also is a significant twist in the relative positions of the two direct
repeats of TBP (Kim and Burley, 1994). In solution, TBP appeared to interact with DNA
in both orientations, with the entries of TFIIA and TFIIB having little effect on the
polarity of the binding (Cox et al., 1997). This result is surprising since one of the
functions of the TATA is thought to involve in determination of polarity with respect to


46
protoplast transient expression systems have been successfully used to study the activity
of a growing number of transcription factor proteins in plants (Goff et al., 1992; Marrs
et al., 1993; McCarty et al., 1991; Yanagisawa and Sheen, 1998). In this study, TFIIB
binding mutants of AtTBP2 were over-expressed in maize suspension cells, and the
effects of these mutants on transcription were determined by monitoring reporter gene
activities.
Materials and Methods
Mutagenesis for AtTBP2
The Altered Site II mutagenesis system of Promega was used to generate amino
acid substitution mutations for AtTBP2 in the Ex-1 vector by the manufacturers
protocol. The targeted residues were El44, El46 and K197. These three amino acids are
predicted to be involved in three charge-charge interactions between TBP and TFIIB in
the TATA-TBP-TFIIBc ternary complex based on the structure determination of the
AtTBP2/hTFIIB complex (Nikolov et al., 1995). Residues El44 and El46 are located in
the C-terminal stirrup, and K197 is located at the C-terminal end of AtTBP2. Each of the
three residues was substituted by an amino acid with the ionic charge opposite to its
wild-type counterpart to form single, double, or triple point-mutations as follows:
E144R, E146R, K197E, E144R/E146R and E144R/E146R/K197E. After confirmation
by DNA sequencing (Microbiology Department Sequencing Core, University of
Florida), both wild-type and mutant TBP cDNAs were released from the Ex-1 vector by
Sal I/BamH I digestion and subcloned into the pBI221 (Clontech) derived plant
expression vector using Sal I/Bgl II sites. The first two residues of TBP within the non-
conserved N-terminal loop were inverted as TM due to subclonings. Both AtTBP2 and


126
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21
that there may be a functional interaction between RAP74 and the conserved region of
TFIIB (Sun and Hampsey, 1995). However, the corresponding mutations in arabidopsis
TFIIB E43R did not show a shift in the start-site of transcription using a hTFIIB-
depleted HeLa nuclear extract in vitro (Baldwin, 1997). It is possible that the residues
involved in start site selection may be species-specific rather than being conserved.
The C-terminal core of TFIIB retains as much capacity to enter a pre-formed
TATA-TBP or TATA-TBP-TFIIA complex as the full-length protein, but blocks further
PIC assembly in vitro (Malik et al., 1993). Consequently, the core domain is
functionally defective in both basal and activated transcription (Malik et al., 1993), and
squelches the function of wild-type TFIIB probably due to the competition for binding
to TBP. Although Pol II is able to interact with the C-terminal end of the second repeat
of the core as has been shown in a pull-down assay (Ha et al., 1993), this interaction
apparently is not sufficient to recruit the enzyme into the PIC in vitro.
Given the essential function and high degree of conservation of TFIIB proteins,
one would expect that they might cross-function in different organisms. Indeed, the
arabidopsis TFIIB is able to replace hTFIIB to support AdML-dependent transcription
by HeLa nuclear extract in vitro (Baldwin and Gurley, 1996). A comparison of dTFIIB
and hTFIIB in a Drosophila embryo-derived in vitro transcription system showed the
same function by the two proteins on three core promoters: Adh, Jockey and AdE4
(Wampler and Kadonaga, 1992). These results suggest that TFIIBs are functionally
exchangeable among higher eukaryotic organisms. However, studies in yeast cells
showed contradictory results, in which both hTFIIB and arabidopsis TFIIB failed to
substitute for yeast TFIIB to support cell growth (Baldwin, 1997; Shaw et al., 1996).


40
that the association of TFIID and holoenzyme on the promoter involves many protein-
protein contacts including those between TBP and TFIIB (Buratowski and Zhou, 1993;
Nikolov et al., 1995), TBP and TFIIA (Geiger et al., 1996; Tan et al., 1996), TBP and
the CTD (Usheva et al., 1992), dTAF40 and TFIIB (Goodrich et al., 1993), hTAF80
and TFIIEa (Hisatake et al., 1995), hTAFn80 and RAP74 (TFIIF) (Hisatake et al.,
1995), and hTAFn250 and RAP74 (Ruppert and Tjian, 1995). However, since these
studies were based on interaction assays performed in vitro using isolated components,
the relative importance and strength of each individual interaction in the context of the
PIC is not known. It is unclear whether each of the potential interaction pathways
between TFIID and holoenzyme is critical for activated transcription, or if their relative
importance is context sensitive.
In recent years, the TBP-TFIIB interaction has been studied intensively,
primarily because these two factors are crucial to both basal and activated transcription.
EMSA experiments have shown that the N-terminal region of TFIIB is dispensable for
TBP binding without affecting the affinity of interaction (Hisatake et al., 1993). For this
reason and its lack of defined crystal structure, only the C-terminal core domain of
hTFIIB (aa 112-316; hTFIIBc) was used in the X-ray crystallographic analysis of
protein structure for the TATA-AtTBP2-hTFIIBc ternary complex (Nikolov et al.,
1995). This analysis revealed that TFIIBc recognizes TBP from beneath the TBP-DNA
complex and, together with TBP, forms a partial clamp around the TATA motif (Fig. 2-
1). In addition, TFIIB contacts both ends of the TATAA to further stabilize the complex.
The overall interactions between TBP and TFIIB involve eight residues from TBP
(E131, Y135, Y143, E144, P145, E146, L147 and K197) and twelve residues from


141
Weinzierl. R. O., Dynlacht, B. D and Tjian, R. (1993a). Largest subunit ofDrosophila
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coactivator. Nature 362, 511-517.
Weinzierl. R. O., Ruppert, S., Dynlacht, B. D., Taese, N., and Tjian. R. (1993b).
Cloning and expression ofDrosophila TAFII60 and human TAFII70 reveal
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100
2A); but not to those containing aa 293-316 (lane 10, Fig. 3-2A). These results localized
the second 14-3-3 binding domain of hTFIIB within the region from aa 262 to 292
(compare lane 10 vs. 9, Fig. 3-2A). Consistent with the results obtained by the C-
terminal deletion series (compare lane 3 vs. 2, Fig. 3-2A), deletion of aa 124-261 in the
N-terminal deletion series resulted in significant reduction in binding (compare lane 9
vs. 8, Fig. 3-2A). Both interaction domains are located in the conserved core of hTFIIB
with aa 124 to 202 within repeat 1 and aa 262 to 292 within repeat 2, which roughly
corresponds to domains previously reported to bind VP16 (Roberts et al., 1993) (Fig. 3-
2B).
The VP16/hTFIIB interaction was previously shown to be enhanced by deletion
of hTFIIB, a phenomenon attributed to disruption of the intramolecular interaction
between the N-terminal region and the C-terminal core within hTFIIB (Roberts and
Green, 1994). In the present study, a similar enhancement in binding to 14-3-3 was also
observed resulting from C-terminal and N-terminal deletions of hTFIIB (compare lane 2
vs. 1; lanes 7 and 8 vs. 6, Fig. 3-2A). This deletion dependent increase in binding was
most pronounced after removal of the entire N-terminal region (aa 1-123) of hTFIIB
(compare lane 8 vs. 7, Fig. 3-2A).
Arabidopsis 14-3-3 Protein and VP 16 Show Similarities in Interactions with hTFIIB
To further investigate the similarity between 14-3-3/hTFIIB and VP16/hTFIIB
interactions, an hTFIIB mutant known to affect VP 16 binding and transcriptional
activation in vitro (Roberts et al., 1993) was tested for its potential to interact with 14-3-
3. The hTFIIB mutation is within the El helix of repeat 1 involving substitutions of two
arginines by two glutamic acids, R185E/R193E. Both wild type and the R185E/R193E


Ill
Internal control:
+1
ubiquitin promoter
Reporter:
+1
GAL4 (lOx) CaMV 35S (-46)
Effectors:
+1
CaMV 35S promoter
Gal4 Atl4-3-3 <|>
DBD
+1
CaMV 35S promoter
Gal4
DBD
Figure 3-8. Diagram of constructs used for in vivo transient expression
assays in plant cells. Expression of the GUS reporter gene was driven by the
CaMV 35S minimal promoter containing 10 copies of the GAL4 element
inserted upstream of TATAA. Expression of effectors Gal4 DBD or Gal4
DBD-AU4-3-3 was driven by the wild type CaMV 35 promoter. The
luciferase reporter driven by the maize ubiquitin promoter was used as an
internal control for normalizing GUS activities to transformation efficiency.


127
Ghosh, S Toth, C, Peterlin. B. M.. and Seto, E. (1996). Synergistic activation of
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10
promoter, and transcription should not be very sensitive to the TGTA mutation. In
contrast, transcription by direct tethering of a holoenzyme component to the promoter,
for example, TFIIB, can occur with only TATA but not TGTA motif, because of lack of
stable binding of TFIID to the TGTA. A study conducted in yeast cells has proven that
this is the case, demonstrating that in vivo, the TFIIB-containing complex, presumably
the holoenzyme, and the TAF-containing complex, presumably TFIID, are separate
before joining to the promoter (Gonzalez-Couto et al., 1997).
Functional analysis has shown that the holoenzyme is able to respond to
activators to stimulate transcription (Koleske and Young, 1994). The purified yeast
holoenzyme is required for transcription in vitro activated by the GAL4-VP16 activator,
while the purified RNA polymerase and general transcription factors fail to respond to
the same activator, suggesting that holoenzyme is the functional form of RNA
polymerase II (Koleske and Young, 1994). This holoenzyme-dependent transcription is
thought to occur through recruitment of the holoenzyme by the activator protein to
increase the local concentration of the enzyme on the promoter high enough for its
function. Consistent with this idea, with a low holoenzyme concentration an activator is
required for high level of transcription, but the same level of transcription can be
achieved without an activator when high holoenzyme concentration is used in the in
vitro transcription reaction (Gaudreau et al., 1998). An activator does not further
stimulate transcription when high holoenzyme concentration is used (Gaudreau et al.,
1998). These findings suggest that an activator stimulates transcription by simple
recruitment of the holoenzyme to the promoter without altering the properties of the
complex.


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Beckmann, H., Chen, J. L., T, O. B and Tjian, R. (1995). Coactivator and promoter-
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1677-1684.
Berger, S. L., Cress. W. D., Cress. A., Triezenberg, S. J., and Guarente, L. (1990).
Selective inhibition of activated but not basal transcription by the acidic
activation domain of VP 16: evidence for transcriptional adaptors. Cell 61. 1199-
1208.
Bihn, E. A., Paul. A. L., Wang, S. W., Erdos, G. W., and Ferl, R. J. (1997). Localization
of 14-3-3 proteins in the nuclei of arabidopsis and maize. Plant J. 12, 1439-1445.
Blair, W. S., Bogerd, H. P., Madore, S. J., and Cullen, B. R. (1994). Mutational analysis
of the transcription activation domain of RelA: identification of a highly
synergistic minimal acidic activation module. Mol. Cell. Biol. 14, 7226-7234.
Blanco, J. C., Wang, I. M., Tsai, S. Y., Tsai, M. J., O'Malley, B. W., Jurutka, P. W.,
Haussler, M. R., and Ozato, K. (1995). Transcription factor TFIIB and the
vitamin D receptor cooperatively activate ligand-dependent transcription. Proc.
Natl. Acad. Sci. USA 92, 1535-1539.
Blau, J., Xiao, H., McCracken, S., O'hare, P., Greenblatt, J., and Bentley, D. (1996).
Three functional classes of transcriptional activation domain. Mol. Cell. Biol. 16.
2044-2055.
Bobb, A. J., Eiben, H. G., and Bustos, M. M. (1995). PvAlf, an embryo-specific acidic
transcriptional activator enhances gene expression from phaseolin and
phytohemagglutinin promoters. Plant J. 8, 331-343.
Braselmann, S., and McCormick, F. (1995). BCR and RAF form a complex in vivo via
14-3-3 proteins. EMBO J. 14, 4839-4848.
Brown, S. A., Weirich, C. S., Newton, E. M., and Kingston, R. E. (1998).
Transcriptional activation domains stimulate initiation and elongation at different
times and via different residues. EMBO J. 17, 3146-3154.
Bryant, G. O., Martel, L. S., Burley, S. K., and Berk, A. J. (1996). Radical mutations
reveal TATA-box binding protein surfaces required for activated transcription in
vivo. Genes Dev. 10, 2491-2504.
Buratowski, S., Hahn, S., Guarente, L., and Sharp, P. A. (1989). Five intermediate
complexes in transcription initiation by RNA polymerase II. Cell 56, 549-561.


137
Ryan, M. P., Jones, R., and Morse, R. H. (1998). SWI-SNF complex participation in
transcriptional activation at a step subsequent to activator binding. Mol. Cell.
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Sadovsky, Y., Webb, P., Lopez. G., Baxter, J. D., Fitzpatrick, P. M Gizang-Ginsberg,
E., Cavadles, V., Parker, M. G., and Kushner, P. J. (1995). Transcriptional
activators differ in their responses to overexpression of TATA-box-binding
protein. Mol. Cell. Biol. 15, 1554-1563.
Samuels, M., and Sharp. P.A. (1986). Purification and characterization of a specific
RNA
polymerase II transcription factor. J. Biol. Chem. 261, 2003-2013.
Sauer, F., Wassarman. D. A., Rubin, G. M., and Tjian, R. (1996). TAF(II)s mediate
activation of transcription in the Drosophila embryo. Cell 87, 1271-1284.
Scharf, K. D., Heider, H., Hohfeld, I., Lyck, R., Schmidt, E., and Nover, L. (1998). The
tomato Hsf system: HsfA2 needs interaction with HsfAl for efficient nuclear
import and may be localized in cytoplasmic heat stress granules. Mol. Cell. Biol.
18, 2240-2251.
Scharf, K. D., Rose, S., Zott, W., Schoffl, F., Nover, L., and Schoff, F. (1990). Three
tomato genes code for heat stress transcription factors with a region of
remarkable homology to the DNA-binding domain of the yeast HSF. EMBO J. 9,
4495-4501.
Schultz, T. F., Medina, J., Hill, A., and Quatrano, R. S. (1998). 14-3-3 proteins are part
of an abscisic acid-VIVIPAROUS 1 (VPl) response complex in the Em promoter
and interact with VPl and EmBPl. Plant Cell 10, 837-848.
Schwechheimer, C., Smith, C., and Bevan, M. W. (1998). The activities of acidic and
glutamine-rich transcriptional activation domains in plant cells: design of
modular transcription factors for high-level expression. Plant Mol. Biol. 36, 195-
204.
Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston, D.
M., and Parvin, J. D. (1997). BRCA1 is a component of the RNA polymerase II
holoenzyme. Proc. Natl. Acad. Sci. USA 94, 5605-5610.
Shaw, S. P., Wingfield, J., Dorsey, M. J., and Ma, J. (1996). Identifying a species-
specific region of yeast TF1 IB in vivo. Mol. Cell. Biol. 16, 3651-3657.


23
by substitution mutations in both DNA and protein. The D2 and E2 helices of the second
repeat of the core domain are believed to form the HTH-like motif for recognizing the
DNA major groove of the BRE. A double point-mutation in this motif, V283A/R286A,
inhibits both TFIIB/BRE binding and basal transcription (Lagrange et al., 1998).
Promoters with sequences less similar to the consensus BRE require eight times higher
TFIIB concentration than those with a consensus BRE to support the same level of basal
transcription (Lagrange et al., 1998). The protein-DNA cross-linking experiments
further showed that TFIIB interacts with the same BRE sequence in both the TATA-
TBP-TFIIB and TATA-TFIIB complexes (Lagrange et al., 1998). However, interactions
with the region downstream of the TATA are absent in the binary complex (TATA-
TFIIB) due to a lack of 80 DNA bending downstream of the TATA, which is a normal
distortion of DNA in the ternary complex (Kim and Burley, 1994). The discovery of the
BRE motif as a bona fide element of the core promoter suggests an important role for
TFIIB in promoter activity. Therefore, the recruitment of TFIIB may be a critical step in
transcription by promoters with no or weak BRE elements.
TBP-TFIIB Interaction
The TBP-TFIIB interaction is thought to be a critical step in the process of PIC
assembly. The EMSA experiments have indicated that the interaction is through the
conserved core domains (C-terminal) in both proteins (Hisatake et al., 1993). Detailed
amino acid contacts were further revealed by crystallography for the TATA-TBP2-
TFIIBc ternary complex (Nikolov et al., 1995). Although many amino acids from both
proteins are involved in numerous electrostatic, hydrogen bond and van der Waals
contacts, the primary interaction site is between the El helix of TFIIB and the C-


In memory of my father, Guoxiang Pan


26
apparently is able to bypass the requirement of the TBP-TFIIB interaction. As the final
comparison, VP 16 activated transcription is not affected in yeast expressing TBP or
TFIIB mutants that impair the TBP-TFIIB interaction, although these same mutations
totally block VP16-activated transcription in HeLa cells (Chou and Struhl, 1997; Tansey
and Herr, 1997). Clearly, different organisms have differential preference for which
interactions (TBP-TFIIB, TBP-CTD, TFIIB-TAF, and so on.) to be used in the PIC
assembly and transcriptional activation. In addition, this preference can be activator-
dependent, as shown by Spl.
Interactions of TBP and TFIIB with Transcriptional Regulatory Proteins
Interaction with Activators
General transcription factors TBP and TFIIB have been reported to be the targets
of PIC recruitment for many activator proteins with different types of activation
domains, including acidic, glutamine-rich and proline-rich domains. The acidic
activators VP 16 and GAL4 interact with both TBP and TFIIB (Kim et al., 1994a;
Roberts et al., 1993; Wu et al., 1996). Deletion analysis for hTFIIB protein has shown
that VP 16 interacts in vitro with TFIIB at the C-terminal ends of the both core repeats,
involving the two helices El and E2, respectively (Roberts et al., 1993). Amino acid
substitution mutations within the El helix greatly inhibit the binding to VP 16 (Roberts
et al., 1993). The bulky hydrophobic residue F442 in the VP 16 activation domain was
shown to be critical for both TFIIB binding (Lin et al., 1991) and transcriptional activity
(Cress, and Triezenberg, 1991), providing an excellent correlation between the potential
for TFIIB recruitment and transcriptional activation. The interactions of TBP with the
acidic activation domains are also highly specific, as demonstrated by the loss of ability


16
the transcription start site. This controversy in the TBP-DNA interaction between in
crystal and in solution has not been resolved.
There are several interesting features about the TBP-DNA interaction. First,
DNA bending has been proven very important for TBP affinity. When circular, instead
of linear, DNA is used, the TBP-TATA interaction is greatly affected by the location of
the TATA element. TBP binds to TATA very tightly when it is located in a position
where there is a slight bend toward major groove. The binding affinity is 100-fold
greater than binding to an unbent linear TATA (Parvin et ai, 1995). Second, TBP seems
to have nonspecific DNA affinity, although it is commonly thought of as the TATA-
specific DNA binding protein. The nonspecific binding of TBP to random DNA
sequences was evidenced by DNase I footprint and detailed kinetic analysis (Coleman
and Pugh, 1995). However, the average affinity of TBP for a random DNA sequence is
about 103 lower than the specific affinity for the TATA motif, and TBP appeared to
translocate along the DNA molecule (Coleman and Pugh, 1995). Therefore, these
authors believed that TBP is able to slide on a DNA template to reach the TATA
element to form stable binding. This model of the TBP-TATA interaction is similar to
that exhibited by the a factor in bacterial transcription (McClure, 1985). Since TBP
contains the amino acid sequences homologous to the a factor (Horikoshi et al., 1989),
it is not surprising to see that they may have a similar mode in interacting with DNA. In
eukaryotes, however, this random scanning of DNA must be strongly directed and
restricted by recruitment mechanisms. Finally, the major groove of the TATA element
seems to also have functional roles in transcription, although it does not interact with
TBP directly. The modification of the major groove by the substitutions of I:C base pair


9
Another striking feature of TFIID is that some TAFs are homologs of histone
proteins. dTAF42 and hTAF31 are homologous to H3; dTAF62 and hTAF80 are
homologous to H4; whereas, dTAF30a/22 and hTAF20/15 are putative H2B homo logs
(Burley and Roeder, 1996). H2A homo logs, however, are still lacking. The fact that
some TAFs are histone-like proteins suggests that TAF proteins may play role(s) in
nucleosome remodeling to facilitate TFIID binding to the core promoter. This possibility
is strongly supported by the cocrystal structure of two Drosophila TAF proteins, in
which the dTAF42/dTAFll62 heterotetramer very much resembles the histone octamer
core structure (Xie et al., 1996). This mimic to the histone core may allow TFIID to
compete with true histones for binding to a chromosomal promoter.
The Holoenzyme
The eukaryotes RNA polymerase II is believed to exist in cells in a complex
with its many associated factor proteins (Kim et al., 1994; Ossipow et al., 1995; Wilson
et al., 1996). This holoenzyme complex is the functional form of RNA polymerase II
and probably contains all activities necessary for a chromosomal gene transcription
except that it lacks the TATA-box binding function. The holoenzyme probably has
several forms, since its composition appears to be different from one prep to another
(Cho et al., 1997; Ossipow et al., 1995; Scully et al., 1997). In general, TFIIB is
considered part of the holoenzyme, but TFIID exists as a separate complex before
forming the PIC with holoenzyme on the core promoter. This was tested by the mutation
of TATA motif, TGTA, to distinguish whether activation of transcription is through
recruitment of TFIID or the holoenzyme. Direct tethering of a TFIID component, a TAF
protein, to the promoter, for example, can artificially localize TFIID complex to the


52
pH 7.5, 0.1 M KC1, 5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 10% glycerol and 0.05%
NP-40.
For protein purification, the cells were disrupted by four one minute bursts of a
probe sonicator, then centrifuged for 30 min at 12,000 rpm. Soluble proteins in the
supernatants were purified with glutathione-agarose beads (Pharmacia) at 4C for 1 hr
with continuous rotation. The beads were pelleted by brief microcentrifugation, then
washed with cold lx protein binding buffer plus 0.5 M KC1 for 10 min, followed by a
final wash with cold lx protein binding buffer, and then suspended in the same buffer.
The quantity of each individual GST fusion protein was determined by comparison to
BSA standard protein in Coomassie stained sodium dodecyl sulfate (SDS)-PAGE.
In vitro Protein Translation
The pGEM-3z vector (Promega) was engineered to have the T7-epitope coding
sequence directly fused with the start codon. Coding sequences of AtTFIIB and AtTBP2
or its mutants were cloned into the modified pGEM-3z vector to express the T7-tagged
proteins in vitro. T7-TFIIB was expressed by a transcription/translation coupled (TNT)
wheat germ system, and T7-TBP was expressed by the rabbit reticulocyte TNT
(Promega), using the manufacturers protocol except that TBPs were expressed at room
temperature.
In vitro GST Pull-Down Assay
To examine the AtTBP2-AtTFIIB interactions, T7-TBP or mutants were
incubated with bead-immobilized GST-TFIIB (15 pg), or reciprocally, T7-TFIIB with
equal amount (15 pg) of GST-TBP or its mutants (the total amount of beads was kept
the same for each binding reaction by adding buffer washed blank glutathione beads


118
which suggests that there were very few functional Gal4 DBD/14-3-3 dimers present to
compete for Gal4 DBD/ftzQ DNA binding at the promoter. Therefore, the low
transcriptional activities shown by the 14-3-3 protein may be due to low levels of
functional Gal4 DBD/14-3-3 proteins in the cells, not to a lack of activation potential.
Finally, low activities may be the result of a combination of low expression
levels and low transcriptional activation potential. The potentially weak activity of Gal4
DBD/14-3-3 may be attributed to a non-processive mode of activation. Like other weak
activators (Blau et al., 1996), the 14-3-3 protein may enhance transcriptional initiation
but not elongation. In this case, transcription of the GUS reporter gene would be
repeatedly aborted to form a series of prematurely terminated transcripts, which are not
reflected by GUS enzyme activity. Primer extension or nuclear run-on assays offer
alternative approaches to examine non-processive transcription (Blau et al., 1996).
Although the 14-3-3 protein was shown to contain two TFIIB binding domains
by deletion analysis, helices-2 and -3 appeared not to be as important as helix-7. In the
context of the full-length protein, a single point mutation within helix-7 almost
abolished binding with TFIIB and TBP, suggesting that helices-2 and -3 make little
additional contribution to interactions between the intact 14-3-3 and TFIIB or TBP.
Since 14-3-3s dimerize through the N-terminal domain, the interacting site within
helices-2 and -3 for TBP and TFIIB may be masked by dimerization. Helix-7 is part of
the box-1 domain (helices-7 and -8) and has been shown as the essential structural motif
for interactions with many proteins, including TPHase (Ichimura et al., 1995), Raf-1 and
Bcr (Ichimura et al., 1997). However, from these previous studies it is not clear whether
both helices of box-1, or only one of them, was required for interactions (Ichimura et al.,


28
molecule with tight folding between the N- and C-terminal domains. The binding of
VP 16 to TFIIB disrupts the intramolecular interaction to expose the buried surfaces of
TFIIB so that it has more surfaces available for other protein-protein interactions. At the
same time, VP 16 experiences a transition from a loose to an ordered structure when it is
complexed with TBP or TFIIB (Shen et al., 1996). The induced structure of VP 16 may
further stabilize its interactions with target proteins within the PIC. It is unclear how
important conformational alterations both in the target proteins and in the activation
domains seen in vitro are in living cells; however, the possibility that conformational
alterations play a major role must always be considered in discussion of activated
transcription.
To further investigate the roles of recruitment of TBP and TFIIB in PIC
assembly and transcriptional activation, many studies have been carried out in yeast and
human cells with an artificial recruitment approach (Chatteijee and Struhl, 1995;
Gonzalez-Couto et al., 1997; Klages and Strubin, 1995; Lee and Struhl, 1997; Xiao et
al., 1997; Xiao et al., 1995b). Instead of being recruited by an activation domain, TBP
or TFIIB is translationally fused directly to a DNA binding domain and forced to
localize to the promoter when the attached DNA binding domain is bound to its
recognition site. This system provides for pure recruitment of only one factor, either
TBP or TFIIB, to directly address whether or not this simple recruitment can lead to PIC
assembly evidenced by transcriptional activation. The results obtained from these
studies have demonstrated that the simple recruitment of either TBP or TFIIB alone is
sufficient to facilitate PIC assembly on the promoter resulting in the activation of
transcription. Activation of transcription by fusing a DNA binding domain to TBP or


41
Figure 2-1. TBP-TFIIB-DNA ternary complex (Nikolov et al., Nature 377:119-
128, 1995), showing location of amino acids residues involved in close contacts
between TBP and TFIIB. Mutations were placed in arabidopsis TBP2 at E144R,
E146R and K197E in this study.


83
factors documented in vitro could serve this bridging function, the TBP-TFIIB
association clearly plays a major role in joining the two complexes during
transcriptional activation in plant cells. This conclusion is based on the TATAA-TBP
and TGTAA-TBPm3 coupled transcription systems in which activated transcription was
most severely inhibited by the E144R/E146R mutation of AtTBP2 for all five activation
domains tested.
The roles of the two individual glutamic acids in the C-terminal stirrup of TBP
appear to differ between in vivo and in vitro transcription systems. Residue El44 was
equally, or probably more, important than El46 in binding TFIIB in vitro as shown in
Figs. 2-4 and 2-5. However, in activated transcription in vivo using an assortment of
activation motifs, El46 was much more critical than El44 (Figs. 2-7 through 2-11, and
2-13 through 2-15). It seems that the degree of involvement in TFIIB binding in vitro
does not strictly correlate with the importance of a particular residue in supporting
activated transcription in vivo. Unlike the direct interaction in vitro in an isolated
system, the in vivo TBP-TFIIB interaction takes place in the context of the PIC in
association with the TATAA motif of the promoter. This interaction is probably much
more complicated than that observed in vitro in terms of possible influences from other
factors including the TATAA, the PIC components, and the activator. In fact, in the
crystal structure of the TATAA-AtTBP2-hTFIIBc ternary complex, E146 is apparently
more involved in TFIIB binding than El44 (Nikolov et al, 1995). Therefore, it is
possible that the relative strength of these two residues in TFIIB binding is different in
the presence and absence of the TATAA motif. Indeed, TBP shows some
conformational distortion after binding to the DNA (Kim and Burley, 1994). This


20
seems to fold onto the core domain through intramolecular interactions to form the
closed conformation (Roberts and Green, 1994). TFIIB with this conformation may
exist in a functionally inactive state. The interaction with an activation domain such as
VP 16 can disrupt the intramolecular interaction of TFIIB to induce the open
conformation that is believed to be in the functionally active state (Roberts and Green,
1994). However, a word of caution must be interjected with respect to the biological
significance of the closed and open states since these studies were conducted in vitro
and TFIIB seems to function in vivo only when associated with the holoenzyme.
TFIIB is a crucial protein in polymerase II-dependent transcription. The deletion
analysis of hTFIIB reveals that almost the entire molecule is required for basal
transcription in vitro (Ha et al., 1993; Hisatake et al., 1993). This seems reasonable
since both the N-terminal domain and the C-terminal core have important roles in the
assembly of the PIC (Ha et al., 1993). The zinc-ribbon in the N-terminal domain of
TFIIB is responsible for the interaction with the small subunit RAP30 of TFIIF
(Buratowski et al., 1989; Ha et al., 1993). The binding of TFIIB to TFIIF seems likely to
be critical in joining TFIIB to the holoenzyme. The single point-mutation C37S in this
domain can disrupt the interaction with RAP30 (Buratowski et al., 1989). For the
adjacent conserved region, one known function is to determine the transcription start-
site. Mutations in this region at E62K, R78C, or E62K/R78C, in yeast sua7 (the TFIIB
gene) showed a downstream shift of the start-site for transcription of the eye l gene
(Pinto et al., 1994). This mutant phenotype can be reversed by a second mutation in the
large subunit RAP74 of TFIIF (SSU71 gene) which restores the normal pattern of
transcriptional initiation of the cycl gene (Sun and Hampsey, 1995). This result suggests


119
1997; Ichimura et al., 1995). The results obtained by this study have shown that helix-8
of box-1 is dispensable in interactions with TFIIB, and isolated helix-7 is capable of
strong binding. The essential involvement of helix-7 in phosphoserine-dependent
(Ichimura et al., 1997) and -independent protein interactions (with components of the
PIC) and its high degree of evolutionary conservation strongly argue that this domain is
critical to 14-3-3 function.
Helix-7 shows amino acid sequence similarity with acidic activators, a
ubiquitous class of transcription factors. Alanine substitution mutagenesis revealed the
importance of the bulky hydrophobic residue, phenylalanine, within helix-7. For several
transcriptional activators including VP 16, phenylalanines within the activation domains
are critical for both transactivation and interactions with general transcription factors
including TFIIB and TBP (Blair et al., 1994; Ingles et al., 1991; Nerlov and Ziff, 1995).
Phenylalanine 186 may play a similar role in interactions between 14-3-3s and general
transcription factors, since the F186A/S187A mutation showed the most severe
disruption of interactions with TFIIB and TBP. Although these experiments can not
distinguish the relative importance between FI86 and SI87, there are no reports of
serine participation in acidic motifs. While FI86 tentatively appears to be the most
critical residue, FI 89 does not seem to be as important for interactions as indicated by
the less severe disruption in binding resulting from the V188A/F189A mutation.
Differential importance for phenylalanines has been previously observed for the VP 16
activation domain, which contains five phenylalanine residues with F442 being the most
critical (Cress and Triezenberg, 1991).


35
457-490. Each of the two subdomains is able to independently activate transcription
(Lyons and Chambn, 1995). Mutational analysis of the activation domain indicates that
the bulky hydrophobic residues are more important than the acidic residues for
transcription (Regier et al., 1993). While some activation domains stimulate
transcription only at the initiation step, VP 16 can activate transcription at both initiation
and elongation stages (Brown et al., 1998). The critical residue F442 appears to only be
important for initiation with the other residues involved in a postinitiation step. These
two activities of VP 16 have been shown to function synergistically in transcription in
vivo (Ghosh et al., 1996). The strong transcriptional activation elicited by VP 16 is
thought to be related to its ability to interact with and recruit multiple general
transcription factors including TBP (Kim et al., 1994a), TFIIB (Roberts et al., 1993),
TFIIA (Kobayashi et al., 1995), TFIIH (Xiao et al., 1994), TAFs (Goodrich et al.,
1993) and PC4 (Ge and Roeder, 1994). Consistently, it is not surprising that high levels
of VP 16 protein expression result in transcriptional squelching, since numerous target
proteins may be sequestered in nonfunctional complexes (Berger et al., 1990; Natesan et
al., 1997).
Ftz
Ftz is a Drosophila transcriptional activator protein involved in segmentation
development during embryogenesis (Kuroiwa et al., 1984), and is comprised of 413
amino acid residues. The N-terminal half of the protein contains the homeodomain,
which is similar to the HTH DNA binding domain of prokaryotic DNA-binding proteins
(Desplan et al., 1988). This domain recognizes the 5-CC/AATTA-3 motif in the
promoters of the segmentation genes such as engrailed (en). The C-terminal half


42
TFIIB (Y165, R169, T176, F177, K188, G192, F195, N207, L208, D243, S249 and
P250). Four out of eight residues from TBP are located in the C-terminal stirrup and
include the following: El44, PI45, El46 and LI47. These four stirrup residues make
contacts with ten amino acids of hTFIIBc to form two salt bridges, two H-bonds, and
seven van der Waals interactions in the ternary complex. Residue El46 makes the most
contacts with TFIIB by forming the strongest salt bridge, two H-bonds and one van der
Waals interaction. It seems likely from this structural information that aa El46 of
arabidopsis TBP2 is the most critical residue for TFIIB binding.
Protein sequence comparisons suggest that the TBP-TFIIB interaction is highly
conserved among eukaryotic organisms. The sequence for the C-terminal stirrup of TBP
is identical among arabidopsis (Kim and Burley, 1994), human (Kao et al., 1990),
Drosophila (Muhich et al., 1990) and yeast (Horikoshi et al., 1989) proteins. Out of the
twelve TBP binding residues of hTFIIBc, all are conserved in dTFIIB, ten in AtTFIIB,
and eight in yTFIIB (Baldwin and Gurley, 1996). The importance of the C-terminal
stirrup of TBP in TFIIB interaction has been confirmed by mutational analysis of hTBP
and yTBP. In hTBP, substitution of E284, E286, or L287 (corresponding to El44, El46
and LI47, respectively, in AtTBP2) by alanine reduces the affinity for TFIIB to about
5% of the wild type level in vitro (Tang et al., 1996). The same alanine substitutions of
yTBP result in 100-, 50-, and 10-fold reductions in TFIIB binding, respectively (Lee and
Struhl, 1997). These stirrup mutations specifically disrupt the interaction of TBP with
TFIIB, but do not affect those with TFIIA, TFIIF, Pol II, TFIIE and TFIIH (Tang et al.,
1996). Although similar studies have not been done in Drosophila or in plants, one


124
Chou, S., and Struhl. K. (1997). Transcriptional activation by TFIIB mutants that are
severely impaired in interaction with promoter DNA and acidic activation
domains. Mol. Cell. Biol. 7 7. 6794-6802.
Christensen. A. H.. and Quail, P. H. (1996). Ubiquitin pro mo ter-based vectors for high-
level expression of selectable and/or screenable marker genes in
monocotyledonous plants. Transgenic Res. 5, 213-218.
Coleman, R. A., and Pugh, B. F. (1997). Slow dimer dissociation of the TATA binding
protein dictates the kinetics of DNA binding. Proc. Natl. Acad. Sci. USA 94,
7221-7226.
Coleman. R. A., and Pugh, B. F. (1995). Evidence for functional binding and stable
sliding of the TATA binding protein on nonspecific DNA. J. Biol. Chem. 270.
13850-13859.
Coleman. R. A., Taggart. A. K., Benjamin, L. R.. and Pugh, B. F. (1995). Dimerization
of the TATA binding protein. J. Biol. Chem. 270, 13842-13849.
Colgn, J., Ashali, H.. and Manley, J. L. (1995). A direct interaction between a
glutamine-rich activator and the N terminus of TFIIB can mediate transcriptional
activation in vivo. Mol. Cell. Biol. 15, 2311-2320.
Colgn, J., Wampler, S., and Manley, J. L. (1993). Interaction between a transcriptional
activator and transcription factor IIB in vivo. Nature 362, 549-553.
Comai, L., Taese, N., and Tjian, R. (1992). The TATA-binding protein and associated
factors are integral components of the RNA polymerase I transcription factor,
SL1. Cell 68, 965-976.
Corden, J. L., Cadena. D. L.. Ahearn, J. M. Jr., and Dahmus, M. E. (1985). A unique
structure at the carboxyl terminus of the largest subunit of eukaryotic RNA
polymerase II. Proc. Natl. Acad. Sci. USA 82, 7934-7938.
Cormack, B. P and Struhl, K. (1992). The TATA-binding protein is required for
transcription by all three nuclear RNA polymerases in yeast cells. Cell 69, 685-
896.
Cortes, P., Flores, O., and Reinberg, D. (1990). Factors involved in specific transcription
by mammalian RNA polymerase II: purification and analysis of transcription
factor IIA and identification of transcription factor II J. Mol. Cell. Biol. 12, 413-
421.
Cote, J.. Quinn, J., Workman, J. L and Peterson, C. L. (1994). Stimulation of GAL4
derivative binding to nucleasomal DNA by yeast SWI/SNF complex. Science
265, 53-60.


CHAPTER 3
THE SPECIFIC INTERACTIONS WITH TBP AND TFIIB IN VITRO SUGGEST
14-3-3 PROTEINS MAY PARTICIPATE IN THE REGULATION OF
TRANSCRIPTION WHEN PART OF A DNA BINDING COMPLEX
Introduction
The 14-3-3 family of proteins was initially characterized by Moore and Perez
(1967) as acidic, soluble proteins which are highly abundant within bovine brain tissues.
Now approximately fifty 14-3-3 genes have been identified from animals, plants and
yeast by researchers in very different fields of study (Ferl, 1996; Wang and Shakes,
1996). In many cases, there are multiple 14-3-3 genes coding for different iso forms,
which are ubiquitously expressed in different cell types. As many as ten 14-3-3 iso forms
have been identified from arabidopsis (Wu et al., 1997b). Sequence comparison of 14-3-
3 cDNAs indicates that 14-3-3 proteins are highly conserved and widely distributed
phylogenetically (Wang and Shakes, 1996). X-ray crystallographic studies indicate
nearly identical structures between two isoforms, suggesting that all isoforms have very
similar structural features (Liu et al., 1995; Xiao et al., 1995a).
While the sequence and structure of the 14-3-3 protein family are highly
conserved, their biological functions are highly diversified in different systems. The 14-
3-3 proteins have been commonly recognized as regulators of a series of kinases
important in multiple signaling pathways, including Raf (Freed et al., 1994; Irie et al.,
1994; Li et al., 1995), Ras (Gelperin et al., 1995; Rommel et al., 1996), Bcr
(Braselmann and McCormick, 1995; Reuther et al., 1994) and PKC (Dellambra et al.,
88


11
The mediator fraction of the holoenzyme is tightly associated with the CTD
domain of polymerase II and consists of some twenty polypeptides including SWI/SNF
(Wilson et al., 1996) and SRB (suppressor of RNA polymerase CTD deletion mutants)
proteins (Chao et al., 1996; Kim et al., 1994b). The SWI/SNF proteins have important
functions in remodeling the chromatin structure. These proteins are able to facilitate
binding of both an activator protein and the PIC components to the promoter DNA in
vitro by disrupting the nucleosomes (Cote et al., 1994; Imbalzano et al., 1994a).
Consistent with their roles in nucleosome disruption, the SWI/SNF proteins were shown
to have high DNA binding affinity and ATPase activity (Quinn et al., 1996; Richmond
and Peterson, 1996). The properties of DNA binding activity are similar to those
exhibited by HMG-box containing proteins. The complex recognizes the minor groove
of the DNA helix and is able to interact with the synthetic four-way junction DNA
(Quinn et al., 1996). Nucleosome disruption is an ATP-dependent process. The ATPase
domains of the SWI/SNF proteins hydrolyze ATP when stimulated by DNA binding.
This ATPase activity appears to be critical to SWF/SNF function, because point
mutations introduced into the ATPase domains resulted in a dominant negative
phenotype in yeast cells (Richmond and Peterson, 1996). The functional importance of
chromatin remodeling by SWI/SNF in transcriptional activation has been shown by
several studies in which a nucleosomal promoter became more accessible to both
upstream activators and PIC components in the presence of SWI/SNF proteins to result
in high levels of transcription in vivo (Bums and Peterson, 1997; Ryan et al., 1998; Wu
and Winston, 1997). Therefore, association of the SWI/SNF proteins with the


85
In contrast to the GAL4-dependent system, the importance of the TBP-TFIIB
interaction in activated transcription is not apparent with the natural complex promoters
such as the CaMV 35S and the ubiquitin promoters. For example, CaMV 35S promoter
activity was strongly stimulated by both wild type and mutant TBP proteins in the
coexpression assays (Fig. 2-16). Moreover, activity of the ubiquitin promoter (internal
control construct) was also not impaired by TBP mutations in these assays (Fig. 2-17).
Therefore, unlike the case with the simple synthetic GAL4 system, TBP stirrup mutants
are, surprisingly, not defective in transcription driven by either of the two natural
complex promoters.
The reduced requirement for a strong TBP-TFIIB interaction shown by the
CaMV 35S and ubiquitin promoters suggests that enhanced stability of the PIC may
result from the involvement of multiple types of activation domains bound to the
upstream elements. The wild type CaMV 35S and GAL4-dependent promoters only
differ in the types and configurations of upstream elements. The core promoter is
identical between the two reporters and is derived from the CaMV 35S minimal
promoter (nt -46). For the GAL4 promoter, transcription is dependent only on the GAL4
element, which in these experiments serves as the binding site for a single type of
activation domain per assay. However, the upstream region of the wild-type CaMV 35S
promoter contains multiple factor binding elements, potentially driven by multiple
activator proteins (Benfey et al., 1990; Lam et al., 1989). One consequence of the
involvement of multiple activator proteins is the possibility that multiple recruitment
pathways may be employed for general transcription factors. This involvement of
multiple interactions in recruitment may stabilize the PIC in a cooperative manner


87
These two observations strongly argue that protein levels in maize cells were similar
between wild type and mutant TBP. The third line of reasoning draws on results from an
analogous system where these same mutations introduced into human TBP did not alter
TBP expression or stability in HeLa cells (Tansey and Herr, 1997). The dramatic
inhibitory effects of AtTBP2 mutations on activated transcription is, therefore, assumed
to reflect the critical roles of the C-terminal stirrup domain of TBP, and support the
conclusion that association between TBP and TFIIB can play a major role in PIC
assembly and activated transcription in plant cells.


84
conformational change may be an important parameter in determining the relative
importance of El 44 and El 46 in TFIIB binding. In this regard, the in vivo results
showing El46 to be more important than El44 in supporting activated transcription is
supported by the structural data (Nikolov et al, 1995), but not in good agreement with
the in vitro GST pull-down results, perhaps due to the lack of DNA in the in vitro
complex.
The functional importance of the TBP-TFIIB interaction in supporting activated
transcription in vivo varies between plants, humans and yeast. Transcription activated by
several different activation domains, including VP 16, is completely independent of the
TBP-TFIIB interaction in yeast cells (Chou and Struhl, 1997; Lee and Struhl, 1997), but
totally dependent on this connection in HeLa cells (Tansey and Herr, 1997). Single
mutations in either of the two glutamic acid residues totally eliminate VP 16 activity in
the TGTAA/TBPm3 coupled system in HeLa cells (Tansey and Herr, 1997), suggesting
that both residues are absolutely required in activated transcription. In plants, however,
these two residues appear to be partially redundant in function, showing proportional
inhibition on transcription by single and double mutations using an analogous
TGTAA/TBPm3 coupled system (Figs. 2-13 through 2-15). Therefore, the relative
importance of El 44 and El 46 of TBP in living cells apparently differs between plants
and humans, although transcriptional activation in both systems requires direct contact
between TBP and TFIIB. In this respect, the pattern of dependence of activated
transcription on the TBP-TFIIB interaction in vivo exhibited by plant transcription
seems to position plants closer to humans than to yeast.


117
localized to the promoter; however, no evidence for transcriptional activity was
observed when Gal4 DBD/14-3-3 was expressed in maize suspension cells.
A possible reason for the lack of activity in maize suspension cells and low
activity in onion epithelial cells is that these particular cell types may lack additional
factors required for efficient activation of 14-3-3-dependent transcription, or these cells
may contain molecules that interfere with 14-3-3 activation.
Another explanation for the low activity may be due to low promoter occupancy
by the Gal4 DBD/14-3-3 protein for a number of reasons. First, the Gal4 DBD/14-3-3
protein may not be stable. Second, the Gal4 DBD may not retain full capability to
recognize the GAL4 sites when fused with the 14-3-3 protein. The Gal4 DBD binds to
DNA only in the dimer form (Carey et al., 1989). The 14-3-3 protein also forms a dimer
through its N-terminal dimerization domain. In dimer form, the N-terminal portion of
the 14-3-3 tends to be buried in the interior of the protein rather than associating with
the solvent exposed surface (Liu et al., 1995; Xiao et al., 1995a). In the Gal4 DBD/14-3-
3, dimerization of the 14-3-3 portion of the chimeric protein may interfere with folding
of the N-terminally-located Gal4 DBD. Finally, high levels of endogenous 14-3-3
proteins and the ease of heterodimer formation between iso forms (Wu et al, 1997a) may
cause the transiently expressed Gal4 DBD/14-3-3 protein to dimerize with the
endogenous 14-3-3s instead of forming homodimers able to bind DNA through the N-
terminal Gal4 DBD.
All of these hypothetical scenarios have the same result: few or no Gal4
DBD/14-3-3 is bound to the promoter. This prediction is consistent with the observation
that Gal4 DBD/14-3-3 did not suppress Gal4 DBD/ftzQ activity when coexpressed,


134
Matsui. T., Segall, J., Weil, P. A., and Roeder, R. G. (1980). Multiple factors required
for accurate initiation of transcription by purified RNA polymerase II. J. Biol.
Chem. 255, 11992-11996.
Matto-Yelin, M., Aitken, A., and Ravid, S. (1997). 14-3-3 inhibits the Dictyostelium
myosin II heavy-chain-specific protein kinase C activity by a direct interaction:
identification of the 14-3-3 binding domain. Mol. Biol. Cell 8, 1889-1899.
McCarty, D. R., T., H., Carson, C. B., Vasil, V., Lazar, M and Vasil, I. K. (1991). The
Viviparous-1 developmental gene of maize encodes a novel transcriptional
activator. Cell 66. 895-905.
McClure. W. R. (1985). Mechanism and control of transcription initiation in
prokaryotes. Ann. Rev. Biochem. 54, 171-204.
Melcher, K., and Johnston, S. A. (1995). GAL4 interacts with TATA-binding protein
and coactivators. Mol. Cell. Biol. 15: 2839-2848.
Mermelstein, F., Yeung, K Cao, J., Inostroza, J. A., Erdjument-Bromage, H., Eagelson,
K., Landsman, D., Levitt, P., Tempst, P., and Reinberg, D. (1996). Requirement
of a corepressor for Drl-mediated repression of transcription. Genes Dev. 10.
1033-1048.
Mittal, V., and Hernandez, N. (1997). Role for the amino-terminal region of human TBP
in U6 snRNA transcription. Science 275, 1136-1140.
Mizzen, C. A., Yang, X. J., Kokubo, T., Brownell, J. E., Bannister, A. J., Owen-Hughes,
T., Workman, J., Wang, L., Berger, S. L., Kouzarides, T., Nakatani, Y., and
Allis, C. D. (1996). The TAF(II)250 subunit of TFIID has histone
acetyltransferase activity. Cell 87, 1261-1270.
Moor, B. W., and Perez, V. J. (1967). Specific acidic proteins of the nervous system. In
Physiological and Biochemical Aspects of Nervous Integaration, ed. FD Carlson.
343-359, Woods Hole, MA: Prentice Hall.
Moqtaderi, Z., Bai, Y., Poon, D., Weil, P. A., and Struhl, K. (1996). TBP-associated
factors are not generally required for transcriptional activation in yeast. Nature
383, 188-191.
Morgan. A., and Burgoyne, R. D. (1992). Exol and Exo2 proteins stimulate calcium-
dependent exocytosis in permeabilized adrenal chromaffin cells. Nature 355,
833-836.
Muhich, M. L., Iida, C. T., Horikoshi, M., Roeder, R. G., and Parker, C. S. (1990).
cDNA clone encoding Drosophila transcription factor TFIID. Proc. Natl. Acad.
Sci. USA 87, 9148-9152.


18
the CTD of the largest subunit of polymerase II (Usheva et al., 1992). These interactions
may be important in facilitating PIC assembly and stabilizing the TBP-TATA complex
as well. In vitro electrophoretic mobility shift assays (EMSA) have shown that TFIIA
and TFIIB can each independently retard the TBP-DNA complex, indicating the
formation of a ternary complex (Imbalzano et al., 1994b). A much greater proportion of
the DNA probes is shifted by each ternary complex than that by the TBP-TATA binary
complex. This observation is interpreted as an indication that TFIIA and TFIIB are able
to fiirther strengthen the DNA binding of TBP. In the TATA-TBP-TFIIA-TFIIB
quaternary complex, TFIIA interacts with N-terminal stirrup of TBP on one side, and
TFIIB interacts with the C-terminal stirrup of TBP on the other side. Physically, TBP,
TFIIA and TFIIB together form a cylindrical clamp around the double-helix of the
TATA DNA (Lagrange et al., 1996). When either TFIIA or TFIIB binds to TBP in a
complex with the DNA, an extended protection of promoter DNA can be observed by
the DNase I footprinting (Malik et al., 1993; Yokomori et al., 1994). It is believed that
both TFIIA and TFIIB also are able to interact with DNA due to their physical proximity
to DNA when bound to TBP. A UV cross-linking study on the TATA-TBP-TFIIA-
TFIIB quaternary complex has confirmed the DNA binding activity for both TFIIA and
TFIIB (Lagrange et al., 1996). Therefore, the enhanced stability of the association of
TBP with DNA in the presence of TFIIA or TFIIB is likely due, at least in part, to DNA-
TFIIA and DNA-TFIIB interactions. Interaction of TBP with the underphosphorylated
CTD of polymerase II may be important in recruiting the holoenzyme to the promoter,
but once assembled, it appears to be inhibitory to promoter clearance for transcriptional
elongation, resulting in paused polymerases on the promoter (Giardina et al., 1992).


107
123456789 --helices
I 1 I I I I I I 14-3-3
Dimerization domain Box-1 domain
binding to GST-hl4-3-3 u deletion mutants
5% input
GST a 3,4 a 1,2 a 5,6,7,8 a 7,8,9 a 1,2,3,4 a 5,6,7,8,9
1 2 3 4 5 6 7
5%
input GST a7 a8
hTFHB-T7
14 15 16
Figure 3-6. Identification of hTFIIB binding domains within hl4-3-3 u. A
series of 14-3-3 deletion mutants were generated by PCR-based cloning such
that individual a-helices were kept intact. Mutant proteins were immobilized
as GST fusions and incubated at 4C for 3 hr with T7-hTFHB in binding
buffer containing 0.15 M KC1. Bound T7-hTFIIB was detected on western
blots using anti-T7 antibody. Regions of hTFIIB interacting with hi 4-3-3 u
were located on helices-2 and -3 (lane 10) and helix-7 (lane 15).


FUNCTIONAL ANALYSIS OF ARABIDOPSIS GENERAL
TRANSCRIPTION FACTORS TBP AND TFIIB
By
SONGQIN PAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1999


GUS/LUC
113
A.
B.
Maize suspension cells
DBD DBD- DBD-
14-3-3 ftzQ
Gal4 DBD fusion proteins
Suppression assay
35.0 -i (maize)
4 ng 2 pg 2 pg
2 pg
2 Pg
Gal4-ftzQ
Gal4 DBD
Gal4-14-3-3
coexpression
Figure 3-10. Evaluation of Transcriptional activity of Gal4 DBD-Atl 4-3-3 in
maize suspension cells. (A) Transcriptional activity of Gal4 DBD (DBD), Gal4
DBD-Atl4-33 (j) (DBD-14-3-3), and Gal4 DBD-ftzQ (DBD-ftzQ) in transient
assays using maize suspension cells. Transformations contained 20 pg of effector
DNA, and assay conditions were identical to those described in Fig. 9 except that
cells were incubated for 22 hr after bombardment. (B) Suppression of Gal4 DBD-
ftzQ activity by coexpression of Gal4 DBD or Gal4 DBD-14-3-3. Amounts of
effector DNAs are indicated. Assays were conducted as in (A).


17
for A:T (TITI) does not affect the binding of TATA with either TBP or TFIID (Lee et
al., 1997). However, this modification significantly inhibits in vitro transcription by
crude HeLa nuclear extracts (Lee et al., 1997). Based on this evidence alone, it is
suggested that the major groove of the TATA element may be also involved in
interaction(s) with other transcription factor(s) (Lee et al., 1997).
In addition to class II genes, some tRNA and 5S RNA genes and most promoters
of RNA polymerase III transcribed-genes also contain the TATA element. These TATA
elements can be universally recognized by TBP, which results in transcriptional
initiation by the different polymerases. The question remains that how these TATA-TBP
complexes can be discriminated by the distinct enzymes. One study has shown that the
orientation of the TBP-TATA complex is the key signal distinguishable for polymerase
II and III. Polymerase II uses the Forward TATA template to transcribe DNA; in
contrast, polymerase III uses the Reverse TATA template (5-TTTTTATA-3) in the
same transcription reaction (Wang and Stumph, 1995). This conclusion is supported by
the observations that forward transcription is only inhibited by the polymerase II-
specific-inhibitor a-amanitin but not by the polymerase Ill-specific-inhibitor tagetitoxin,
and transcription from the reverse TATA was inhibited by only tagetitoxin but not a-
amanitin (Wang and Stumph, 1995). These results indicate that the TBP-TATA complex
can function bi-directionally in the same core promoter with the polarity of transcription
determined by the different RNA polymerases.
Besides the important function in recognizing the TATA and association with
TAFs, TBP also interacts with several other components of the PIC, including TFIIA
(Geiger et al., 1996; Tan et al., 1996), TFIIB (Ha et al., 1993; Nikolov et al., 1995) and


92
important targets for those activator proteins with acidic (Kim et al., 1994a; Roberts et
al., 1993), proline-rich (Kim and Roeder, 1994), or glutamine-rich activation domains
(Colgn et al., 1995; Nakshatri et al., 1995). To explore the possible function(s) of the
14-3-3 proteins in transcription, the potential for these proteins to directly interact with
TBP and TFIIB was evaluated in vitro, and their abilities to activate transcription were
assessed in vivo. The 14-3-3 proteins are shown to have specific affinities for TBP,
TFIIB, and hTAFn32 in vitro. In addition, the transiently expressed Gal4 DBD/14-3-3
chimeric protein can activate GAL4-dependent GUS gene expression in plants.
Materials and Methods
Protein Expression in E. coli
The cDNAs encoding either 14-3-3, TFIIB, or TBP were inserted into the
polylinkers of pGEX-2TK (Pharmacia), pET-15b, pET-24b and pET-24d (Novagen) E.
coli expression vectors. The vector pGEX-2TK has the coding sequence for glutathione-
S-transferase (GST) followed by a thrombin-cleavage site and a kinase phosphorylation
site. Following the start codon, the pET vectors have the His- and T7-tag coding
sequence, respectively. In order to maintain the correct reading frame, extra amino acids
were introduced into either the C- or -N-terminal ends of the desired proteins by
subcloning.
For protein expression, pGEX-2TK constructs were transformed into E. coli
strain BL21, and selected by ampicillin resistance. The transformed cells were grown
overnight, then were diluted 1:100 in 50 ml of LB containing 10pg/ml ampicillin, and
grown an additional 3 hr at 37C before IPTG induction. The expression of GST fusion
proteins was induced by 0.01 or 0.1 mM IPTG at 37C for 1 hr. The pET constructs were


136
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mutations defines a domain of TFIIB involved in transcription start site selection
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Puig, O. M., Belles, E., Lopez-Rodas, G., Sendra, R., and Tordera. V. (1998).
Interaction between N-terminal domain of H4 and DNA is regulated by the
acetylation degree. Biochem. Biophys. Acta 1397, 79-90.
Quinn. J., Fyrberg, A. M., Ganster, R. W., Schmidt. M. C., and Peterson C. L. (1996).
DNA-binding properties of the yeast SWI/SNF complex. Nature 379, 844-847.
Regier, J. L., Shen. F.. and Triezenberg, S. J. (1993). Pattern of aromatic and
hydrophobic amino acids critical for one of two subdomains of the VP 16
transcriptional activator. Proc. Natl. Acad. Sci. USA 90, 883-887.
Reuther, G. W., Fu, H., Cripe, L. D., Collier, R. J., and Pendergast, A. M. (1994).
Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3
family. Science 266, 129-133.
Richmond, E., and Peterson, C. L. (1996). Functional analysis of the DNA-stimulated
ATPase domain of yeast SWI2/SNF2. Nucleic Acids Res. 24, 3685-3692.
Roberts, S. G. E., and Green, M. R. (1994). Activator-induced conformational change in
general transcription factor TFIIB. Nature 371, 717-720.
Roberts, S. G. E., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1993).
Interaction between an acidic activator and transcription factor TFIIB is required
for transcriptional activation. Nature 363, 741-744.
Rommel, C., Radziwill, G., Lovric, J., Noeldeke, J., Heinicke, T., Jones, D., Aitken, A.,
and Moelling, K. (1996). Activated Ras displaces 14-3-3 protein from the amino
terminus ofc-Raf-1. Oncogene 12, 609-619.
Ruppert, S., and Tjian, R. (1995). Human TAFII250 interacts with RAP74: implications
for RNA polymerase II initiation. Genes Dev. 9, 2747-2755.


102
1% Input TFIIB
R185E R185E R185E R185E
R193E WT R193E WT R193E WT R193E WT
GST-fusion: VP16A456 VP16 Atl4-3'^
F442P
hTEIIB-T7 tag
(wt or mutant)
Figure 3-3. Comparison of hTFIIB and mutated hTFIEB interactions with
immobilized 14-3-3, VP 16 and mutant VP 16. Free hTFIIB (T7-tagged) or its
mutant protein (R185E/R193E) were incubated at 4C for 1 hr with immobilized
GST-VP16A456F442P (as a negative control), GST-VP16, and GST-14-3-3 in
binding buffer containing 0.15 M KC1. The hTFIIB point mutation R185E/R193E
inhibited interactions with VP 16 and 14-3-3 to a similar degree. Bound hTFIIB was
detected on western blots using anti-T7 antibody.


BIOGRAPHICAL SKETCH
Songqin Pan was bom in Yuhuan County, Zhejiang Province, southeast of
China, on December 5, 1962. His hometown is on an island in the East Sea of China,
which is not far away from Taiwan. After graduation from high school in the summer of
1978. and benefited by the new era of reforming and opening of China after the Cultural
Revolution, he was lucky to be admitted by Zhejiang Agricultural University (now the
College of Agriculture, Zhejiang Univerity) by passing the national tests with which
only one out of thirty participants could be successful. In July 1982 when he completed
the B.S. degree in horticultural science, he came back home to serve as an extension
specialist until the end of 1990. During his service for the local horticultural industry, he
was promoted to be an Associate Director of Agricultural Bureau of the local county in
1985 and to be a professional Agronomist in 1989. In December 1990, he came to the
United States to be a visiting scholar in the Department of Horticulture at the
Pennsylvania State University. After eight months, he was admitted by the Department
of Horticulture at University of Wisconsin-Madison as a graduate student and obtained
an M.S. degree in horticultural science in May 1993. In May 1994, he was enrolled in
the Program of Plant Molecular and Cellular Biology at the University of Florida as a
Ph.D. student and conducted a research project on transcriptional regulation in higher
plants as described in this dissertation.
143


79
mutations, whereas, that driven by VP16-Gal4 AD was less susceptible. The double
mutation E144R/E146R showed an 80% inhibition for the activator ftzQ-Gal4 AD
(compare column 4 vs. 2, Fig. 2-18), but only a 45% reduction in transcription activated
by VP16-Gal4 AD (compare column 8 vs. 6, Fig. 2-18). The differential effects of
stirrup mutants on these two double activation domains reveal an unexpected complexity
in mechanism that must underlie these results. On one hand, the decreased sensitivity to
TBP stirrup mutations exhibited by VP16-Gal4 AD suggests that the presence of
multiple pathways (or contacts) of activation inherent in double activation domains leads
to a diminished dependence on TBP-TFIIB interactions for activated transcription.
However, for ftzQ-Gal4 AD, this model is contradicted since very little decrease in
reliance on TBP-TFIIB interactions was evident. Clearly, the importance of direct TBP-
TFIIB interactions in activated transcription is context sensitive, for reasons that are
poorly understood.
Discussion
The present study is the first in plants to examine the biological function of an
important protein-protein interaction within the PIC, contacts between TBP and TFIIB.
Transient assays in maize cells demonstrated that the TBP-TFIIB interaction can be a
major pathway for activated transcription; however, the functional importance of this
association to the process of transcription is clearly context sensitive in plants. It is
essential for efficient gene expression in certain situations, and in others, contacts
between these two general transcription factors appear to be dispensable. Activated
transcription from a simple promoter driven by a single activation motif is heavily
dependent upon the TBP-TFIIB interaction, whereas in complex natural promoters, the


5
TFIID
TFIID is a complex comprised of TBP (TATA binding protein) and TAFs (TBP
associated factors). TBP is the only protein commonly required for transcription by all
three RNA polymerases in eukaryotic organisms (Cormack and Struhl, 1992). Although
TBP by its own is able to bind the TATA element of the core promoters in vitro, the
specificity of promoter recognition by TBP in vivo is likely determined by TAF proteins.
Distinct TAFs are associated with TBP on the core promoters of different classes of
genes to form SL1 (Comai et al., 1992), TFIID (Timmers et al., 1992) and TFIIIB
(Taggart et al., 1992) complexes to support transcription by Pol I, Pol II and Pol III,
respectively. These TAFs may serve as promoter selectivity factors for different classes
of genes (Jordan et al., 1996; Taggart et al., 1992; Verrijzer et al., 1995), or may
facilitate the recognition by the corresponding polymerase by interactions with its
component proteins, or may provide the targets for the upstream activator proteins in
activated transcription (Beckmann et al., 1995).
The TFIID complex is believed to have several forms. The basic, or general,
form of TFIID presumably functions in all polymerase II transcription, and contains at
least eight different TAFs (Burley and Roeder, 1996). cDNAs for TAFs have been
isolated from human, yeast and Drosophila with predicted protein sizes ranging from
15-250 kD (Burley and Roeder, 1996). In vitro, the proteins expressed by these cDNAs
together with TBP are able to reconstitute the TFIID complex (Chen et al., 1994).
Although there has not been a TAF identified from plants, it has been shown that there
is a TFIID activity in plant cells (Washburn et al., 1997), and several expressed
sequence tags (EST) clones from maize and arabidopsis show sequence similarity to


95
enhanced autoradiography. The lx protein binding buffer contained 20 mM HEPES, pH
7.5, 0.15 M KC1, 5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA and 0.05% NP-40.
Site-Specific Point Mutagenesis
The Altered Site II mutagenesis system by Promega was used to generate alanine
substitution mutants for At 14-3-3 <|> by following the manufacturers protocol. The
poly linker of the pALTER-Exl vector (Promega) was engineered so that the T7-tag
coding sequence (MASMTGGQQMG) was placed immediately after the start codon.
The arabidopsis 14-3-3 cDNA (Wu et al, 1997) was subcloned into the pALTER-Exl
vector (Promega) in frame with the T7-tag coding sequence. All the desired mutants
were confirmed by DNA sequencing (Microbiology Department Sequencing Core,
University of Florida) and subcloned into pGEX-2TK (Pharmacia) for expression in E.
coli.
Transient Expression Assay
Transient assays were conducted with either maize suspension cells or onion
epidermal peels, and the experimental procedures were essentially the same as those
described in the previous chapter of this dissertation with some exceptions reported
below.
The middle layers of large yellow onions obtained from a local supermarket
were cut into 2x2 cm pieces. The epidermal layers then were peeled off and placed on
individual MS plates (Murashige and Skoog, 1962) immediately before use. Each DNA-
gold preparation contained 5 pg of the internal control plasmid (Ubi/LUC) and 5 pg of
the GAL4/GUS reporter plasmid in addition to various amounts of effector plasmid


94
A and T7 tag by immunoaffinity. The pelleting and washes for T7-tagged proteins were
the same as those for GST fusion proteins. The quantity of each individual protein was
estimated by comparison to BSA standard protein in Coomassie stained SDS-PAGE.
In vitro Protein Translation
Human TAFh32 and TAFn55 proteins were obtained by in vitro translation and
labeled by 35S-methionine using a transcription/translation coupled (TNT) rabbit
reticulocyte system according to the manufacturers protocol (Promega). The TAF
cDNAs were cloned into the pET-24d vector (Novagen), which utilizes the T7 promoter.
In vitro GST Pull-Down Assay
To study the interactions of 14-3-3 proteins with the general transcription factor
proteins, one of the two interacting proteins was fused with GST and purified on
glutathione-agarose beads. The bead-immobilized proteins were quantified by
comparing to BSA standards in Coomassie stained SDS-PAGE, and 5 pg of them was
used in each binding reaction. The second protein, from an E. coli lysate or from rabbit
reticulocyte translation, was then incubated with the beads for 1-3 hr at either 4C or
room temperature (described in figure legends) in 600 pi of lx protein binding buffer
containing 0.5% BSA with continuous rotation. Salt concentration was from 0.1 to 0.15
M KC1 as indicated in figure legends. The beads were then washed with lx protein
binding buffer (4 times, 1 ml each). The buffer for the first two washes contained 0.5%
BSA. The beads were pelleted by centrifugation for 15 seconds using a mini-centrifuge
after each washing. The bound protein molecules were finally resolved by SDS-PAGE
and detected by ECL immunoblotting (Pharmacia) or by a 2,5-diphenyl-oxazole (PPO) -


for TFIIB binding, however, mutation E146R showed much more severe inhibition on
activated transcription than E144R in vivo. The natural CaMV 35S and maize ubiquitin
promoters showed independence of TBP-TFIIB interaction for their activities, indicating
that TBP-TFIIB interaction is not required for transcription driven by complex
promoters.
In a second series of experiments, the potential of 14-3-3 proteins to regulate
transcription in plants was evaluated by a combination of in vitro and in vivo studies. In
vitro, 14-3-3 proteins have specific affinity for both TBP and TFIIB as demonstrated by
domain mapping and alanine substitution analysis. 14-3-3 and VP 16 seemed to share the
same or overlapping binding domain within TFIIB, and competed with each other in the
same TFIIB binding reaction. Given the fact that 14-3-3 interacts with several activators
such as VP1, EmBPl and glucocorticoid receptor, 14-3-3 proteins may have role(s) as
transcriptional co-activators to bridge the activators and the PIC. However, the
Gal4DBD/14-3-3 chimera showed a low or no activation potential in plant cells,
probably due to low protein level of the GaMDBD/14-3-3 with appropriate function to
recognize the GAL4 binding sites.


GUS/LUC
58
Basal transcription in maize cells
TBP mutants
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
TBP2
T7
vector
TFIIB
I
E144R
E146R
K197E
E146R
E144R
1 2 3 4 5 6 7 8
Effector constructs coexpressed with the basal transcription
reporter (CaMV 35S minimal promoter/GUS)
Figure 2-6. Effects of coexpression of TBP and TFIIB on basal
transcription in maize cells. Basal transcription of the GUS reporter was
driven by the CaMV 35S minimal promoter (nt -46) without upstream
activation elements. The same amount of DNA (10 pg) was used for each
effector construct. The T7 vector was used to express TBP or TFIIB, and
contains the T7 epitope coding sequence. The construct with the minimal
promoter deleted (ATAT A) served as a control for no transcription, and was
coexpressed with the T7 vector. Transcriptional activity was normalized by
the internal control LUC activity and expressed as arbitrary units of relative
GUS/LUC (nM Mu/cpm). The results of one representative experiment are
shown. The data for each treatment was the average of three replicates.


27
of yTBPLl 14K mutant to interact with VP 16 and GAL4 activation domains (Kim et al.,
1994a; Melcher and Johnston, 1995). In addition, the level of GAL4-activated
transcription in vivo is positively correlated with the affinity in vitro for TBP and TFIIB,
suggesting that the recruitment of TBP and/or TFIIB is critical in the activated
transcription in yeast (Wu et al., 1996). The CTF1 proline-rich activation domain
interacts with TFIIB, but not TBP, to facilitate the entry of TFIIB into the TATA-TBP
complex (Kim and Roeder, 1994). In addition, the glutamine-rich activation domain of
ftzQ interacts with TFIIB in vitro at its N-terminal zinc-ribbon domain. The biological
significance of the /izQ-TFIIB interaction was implied by experiments where deletion of
the C-terminal core of TFIIB severely squelched ftzQ activity in transcription in vivo
(Colgn et al., 1995).
The interaction between an activator protein and TBP or TFIIB not only provides
a mechanism for recruitment of the latter to the core promoter, but also facilitates, in a
reciprocal manner, binding of the activator to its upstream element. These cooperative
effects between the activator binding to an enhancer sequence and PIC assembly on the
core promoter have been observed in several cases, including the hsp70 promoter
(Mason and Lis, 1997), GAL4 (Vashee and Kodadek, 1995), p53 (Chen et al., 1993) and
MyoD (Heller and Bengal, 1998) activators. These studies showed that the bindings of
the activators to the DNA elements were all enhanced by the interactions of the
activators with TBP. Additionally, the interaction between an activator and TBP or
TFIIB can alter the conformations for both interacting proteins. Proteolytic analysis of
the TFIIB protein showed significantly different patterns of protease digestion before
and after binding to VP 16 (Roberts and Green, 1994). In vitro, TFIIB itself is a compact


81
yeast systems, analogous point mutations are much less severe in plants, ranging from
only 2- to 7-fold in the GST pull-down assays. It is not known whether this difference
may be due to differences in the method of assay. For example, is the nature of
association of TBP with DNA in EMSA assays the one that further enhances the
dependence of the TBP-TFIIB interaction on these two glutamic acid residues? Another
consideration is the structural differences between plant and animal TBPs. Compared to
human and yeast TBPs, AtTBP2 lacks the nonconserved N-terminal region whose
function is still unclear with respect to transcriptional regulation.
In maize suspension cells, basal transcription dependent on the CaMV 35S
minimal promoter was specifically stimulated by coexpression of TBP but not TFIIB.
Although basal transcription is normally defined by in vitro experiments using
reconstituted transcription systems, it can occasionally be observed in vivo. For
example, expression of basal transcription in Drosophila L2 cells has been reported
(Colgn et al., 1993). This observation in Drosophila and the results obtained in this
study indicate that basal transcription can also occur in vivo. There are other parallels
between the present study in plants and the Drosophila study. For example, basal
transcription in Drosophila was stimulated by coexpression of dTBP by 20-fold,
whereas dTFIIB was totally ineffective in stimulating basal activity (Colgn et al.,
1993). Although AtTFIIB protein expression in maize cells was not determined in the
present study, it seems to be normal, since preliminary experiments showed that
activated transcription by several GAL4 fused activation domains was stimulated by
coexpression of AtTFIIB (not included in this dissertation). Therefore, it is likely that
the TBP or TFIID concentration on the promoter, rather than TFIIB or holoenzyme, is


77
insensitive to the same TBP mutations. Compared to a simple promoter such as GAL4,
serving as the binding site for a single type of activator, complex natural promoters are
able to minimize the requirement for direct TBP-TFIIB interaction, perhaps due to the
utilization of multiple recruitment pathways made possible by the involvement of
multiple activators.
These results also indicate that TBP was a rate-limiting factor for transcription
driven by the CaMV 35S promoter, but not for the ubiquitin promoter. Similar
differential responses by different activators to over-expression of TBP were previously
observed in HeLa (Sadovsky et al., 1995). The increases in activity of the ubiquitin
promoter seen with the mutant forms of TBP, compared to exogenous wild type TBP,
may indicate that the mutated TBPs are less efficient, but still functional (Fig. 2-17).
This decrease in efficiency is presumably reflected in their reduced capacity to squelch.
A similar effect was seen with the CaMV 35S promoter, where most of the stirrup
mutants showed less activity than exogenous wild type TBP, but nevertheless stimulated
transcription compared to endogenous TBP (T7 vector).
Reliance on Multiple Activation Pathways Can Partially Compensate Suppression by
TBP Stirrup Mutations
To test whether the TBP-TFIIB interaction is still important in transcription
involving multiple recruitment pathways, two activation domains were fused together to
drive GAL4-dependent transcription. The Gal4 AD was fused to two different activation
domains to produce two double activators, Gal4 DBD/VP6-Gal4 AD and Gal4
DBD/ftzQ-Gal4 AD. These were coexpressed in transient assays with wild type or
mutated TBP to evaluate suppression by stirrup mutants. As seen in Fig. 2-18,
transcription activated by ftzQ-Gal4 AD remained sensitive to the TBP stirrup


43
would expect that the C-terminal stirrup of TBP also serves as a critical TFIIB binding
domain during the PIC assembly in these systems as well.
The altered specificity system is a powerful approach used in defining
functional roles of the TBP-TFIIB interaction in human systems. This system is
established by the mutations in both the TATA-box and TBP. This specificity was first
identified in yeast by using the TGTA-his3 gene to select for a TBP mutant that was
able to support cell growth (Strubin and Struhl, 1992). The mutant TBP, TBPm3, has
three substitutions for the residues located in its concave DNA binding surface as
follows: I194F, V203T and L205V. TBPm3 possess an extended DNA binding
specificity from TATAA to TGTAA, to which the wild type TBP is unable to bind as
demonstrated by EMSA experiments (Strubin and Struhl, 1992). Therefore, transcription
of a reporter gene containing the TGTA instead of the TATA motif would be dependent
on TBPm3, but not the endogenous TBP. This system allows precise measurement of
the transcriptional effect of additional mutations on TBPm3 without interference from
the endogenous TBP protein in a transient expression assay. The altered specificity of
TBPm3 has also been confirmed in studies with hTBP (Bryant et al., 1996) and AtTBPs
(Heard et al., 1993).
The effects of the C-terminal stirrup mutations of TBP on transcription were
assessed using this TBPm3/TGTA coupled system. The three single substitution
mutations, E284R, E286R and L287E, of the C-terminal stirrup of hTBP abolished basal
transcription in vitro (Bryant et al., 1996). In addition, E284R and L287E mutations
greatly inhibited activated transcription by VP 16 or El A in COS cells, while the E286R
mutation showed a moderate reduction (about 50%) in transcription (Bryant et al.,


GUS/LUC
62
Transcription activated by Gal4 DBD/Gal4 AD
TBP mutants
Effector constructs coexpressed with Gal4 DBD/Gal4 AD
Figure 2-9. Effects of coexpression of TBP or the C-terminal stirrup mutants
on Gal4 AD transcriptional activity. The details are the same as in Fig. 2-7.


LIST OF ABBREVIATIONS
AD activation domain
AdML adenovirus major late promoter
bp base pair
CaMV cauliflower mosaic virus
Cpm counts per minute
CTD carboxy-terminal domain
DAB a complex of TATA, TBP, TFIIA and TFIIB
DB a complex of TATA, TBP, and TFIIB
DBD DNA-binding domain
EMSA electrophoretic mobility shift assay
Ftz fushi taratzu
GST glutathione S-transferase
GUS/LUC relative P-glucuronidase/luciferase activity
HeLa a human cancer cell line
hr hour
HTH helix-tum-helix
LpHSF8 tomato heat shock transcription factor 8
min minute
ml mililiter
mM milimolar
NMR nuclear magnetic resonance
PCR polymerase chain reaction
PIC the pre-initiation complex
Pol II RNA polymerase II
RAP30 the small subunit of TFIIF
RAP74 the large subunit of TFIIF
sec second
SL1 TBP-containing factor for RNA polymerase I
SRB suppressor of CTD mutations of RNA pol II
TAF TBP-associated factor
TATA core promoter element
TBP TATA-binding protein
TFIIA transcription factor for pol II A
TFIIB transcription factor for pol II B
TFIID transcription factor for pol IID
TFIIE transcription factor for pol II E
TFIIF transcription factor for pol IIF
TFIIH transcription factor for pol II H
vii


56
A. Immobilized GST-TFIIB; TBP free
10% Inputs Binding to GST-AtTFIIB
T7-AtTBP2
& mutants
WT E144R E146R E144R
E146R
WT E144R E146R E144R
E146R
B.
E144R
E146R
E144R
E146R
10% inputs
Bound
WT
E146R
E144R
E144R
E146R
AtTBP2 and mutants (free)
Figure 2-5. Binding of free TBP to immobilized GST-TFIIB. (A) Western blot
of AtTBP2 and mutants bound to immobilized GST-AtTFIIB. Western blot was
probed with anti-T7 monoclonal antibody. T7-AtTBP2 and mutant proteins were
synthesized by a coupled transcription/translation reaction in rabbit reticulocyte.
(B) Quantification of imagines for (A) as discussed for Fig. 2-4.


73
transcription by the C-terminal stirrup mutations was also apparent even if GUS activity
was not normalized by the internal control luciferase activity (Fig. 2-15B). Overall,
these results are parallel to those observed in experiments using the TATAA/TBP
system, although it appears that TBPm3 is much more sensitive to C-terminal stirrup
mutations than TBP. In both systems, residue El46 of TBP appears to be more critical
than El44 in supporting the TBP-TFIIB interaction in vivo, which is consistent with
predictions based on structure (Nikolov et al, 1995).
Complex Promoters Show Much Less Dependence on the TBP-TFIIB Interaction
In addition to testing the synthetic GAL4 promoter driven by a single activation
domain fused to the Gal4 DBD, two natural promoters, the CaMV 35S and maize
ubiquitin promoters, were also evaluated regarding the significance of the TBP-TFIIB
interaction. These two promoters are more complex than the synthetic GAL4 promoter
and rely on multiple activator proteins endogenous to maize cells for activity (Benfey et
al., 1990; Genschik et al., 1994; Hoffman et al., 1991; Lam et al., 1989). The CaMV
35S/GUS and Ubi/LUC reporters were coexpressed with either TBP or its mutants in the
same experiment. The effects of TBP mutations on CaMV 35S and ubiquitin promoter
activities were presented in Fig. 2-16 and Fig. 2-17, respectively. Transcription by the
CaMV 35S promoter in transient assays was strongly stimulated by over-expression of
wild-type TBP and all stirrup mutants (compare columns 2, 3, 4, 5, and 6 vs. 1, Fig. 2-
16A and B), while ubiquitin promoter activity was only minimally affected by
coexpression of TBP and mutants (compare columns 2, 3, 4, 5, and 6 vs. 1, Fig. 2-17). In
contrast to the strongly inhibitory effect of stirrup mutations in transcription driven by
the GAL4 promoter, transcription of these two natural promoters was almost completely


50
GaI4 DBD
fusions:
+1
CaMV 35S promoter GaI4 AD:
DBD
ftzQ
Gal4
LpHSF8
VP1
VP16
T7-tag fusions:
+1
CaMV 35S promoter
T7 effector:
wt AtTBP2
TBP mutants
wt AtTFIIB
Figure 2-3. Effector constructs: Gal4 DBD fusions and T7 epitope-tagged


This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as
requirements for the degree of Doctor of Philosophy.
May, 1999.
fulfillment of the
Dean, Graduate School


104
to competition between GST-VP16 and 14-3-3 for binding to hTFIIB, or competition
between hTFIIB and 14-3-3 for binding to GST-VP16. In the former case, most of the
hTFIIB molecules would be complexed with 14-3-3 molecules in solution and removed
during washing steps. In the latter case, 14-3-3 would bind to GST-VP16 and be
detected by anti-14-3-3 antibody. This possibility was excluded since no additional 14-
3-3 band was seen in the same blot re-probed by anti-14-3-3 antibody (data not shown).
Therefore, it is concluded that the greatly reduced binding of hTFIIB to VP 16 was due
to competition between VP 16 and arabidopsis 14-3-3 for binding to hTFIIB.
In a reciprocal experiment shown in Fig. 3-4B, T7-tagged 14-3-3 was
immobilized on beads containing covalently linked protein A using anti-T7
immunoaffinity as diagramed. Free hTFIIB in a crude bacterial extract was then
incubated with immobilized 14-3-3 in the presence of equal amounts of either GST (lane
2, Fig. 3-4B) or GST-VP16 (lane 1, Fig. 3-4B) in excess. Bound hTFIIB was detected in
a western blot using anti-hTFIIB monoclonal antibody (Promega). The difference in
binding between lanes 1 and 2 (Fig. 3-4B) clearly show that VP 16 strongly inhibited
binding of hTFIIB to 14-3-3, consistent with competition between VP 16 and 14-3-3 for
interactions with hTFIIB.
Human 14-3-3 o Shows Affinity for Human TFIIB, TBP, and TAFn32, but not for
TAF55
Due to the lack of cloned plant TAFns and to avoid possible difficulties in
interpretation resulting from the use of components from plants and humans, homotypic
interactions between human 14-3-3 (hi4-3-3) with human general transcription factors
and co-factors were evaluated. The GST-hl4-3-3 u protein was immobilized on
glutathione beads and incubated with bacterially expressed human TBP and TFIIB, or


13
prevent formation of a functional PIC complex on the promoter. Overall, it seems that
distinct sets of SRBs are used in either transcriptional activation or repression.
General Transcription Factor TBP
The central component of the TFIID complex, TBP, is the first general
transcription factor cloned and characterized. The cDNAs for TBP have been isolated
from many eukaryotic organisms, including animals (Kao et al., 1990; Muhich et al.,
1990), plants (Gasch et al., 1990; Haass and Feix, 1992) and yeast (Horikoshi et al.,
1989). While the TBP gene appears to be single copy in both animals and yeast, there
are at least two copies in plants, including both dicots (arabidopsis) (Gasch et al., 1990)
and monocots (maize) (Haass and Feix, 1992). The known TBP protein sequences show
a high degree of conservation in their C-terminal 180 amino acids, which is the
functional core domain for binding the TATA motif and supporting transcription (Hoey
et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990). In contrast, the N-terminal
region of the proteins is much diverged, and its function is not clear. Deletion of this
region does not affect polymerase II transcription and yeast cell growth, indicating that
it is not important in TBP function. However, inclusion of the antibody mAblC2 against
this region inhibits in vitro transcription from TATA-containing, but not TATA-less
promoters (Lescure et al., 1994). Another study indicates that the N-terminal region of
hTBP down-regulates the binding of TBP with the U6 TATA box of the polymerase III
promoter (Mittal and Hernandez, 1997). These results suggest that the N-terminal region
may have a regulatory role on TBP function. Recently, a tissue-specific TBP homolog,
TRF, was identified from Drosophila (Hansen et al., 1997). Like TBP, TRF possesses
normal function in TATA binding, DB complex formation, and supports both basal and


68
vector (T7 vector) was coexpressed with the TATAA and TGTAA GUS reporters as
shown in columns 1 and 2 of Figs. 2-13 through 2-15. Endogenous activity was much
reduced with the TGTAA reporter. Furthermore, over-expression of wild type
arabidopsis TBP produced only low increases in activity compared to the endogenous
TBP, which is seen by comparing columns 2 and 3 of Figs. 2-13 through 2-15. In
contrast, over-expression of AtTBPm3 resulted in significantly more activity compared
to wild type AtTBP, ranging from a very modest increase in activity for VP1 (Fig. 2-14;
column 4) to a complete restoration of endogenous activity for the tomato heat shock
factor LpHSF8 (Fig. 2-15A; column 4).
The importance of TBP-TFIIB interactions was demonstrated by analysis of the
C-terminal stirrup mutations introduced into the altered specificity system. Mutations
E144R, E146R and E144R/E146R were introduced into AtTBPm3 and assayed using
the TGTAA reporter. When activated transcription relied on exogenous TBPm3, all the
three stirrup mutations suppressed activated transcription (Figs. 2-13 through 2-15).
Unlike in the stimulation seen with TBP and the TATAA reporter, the E144R mutation
inhibited activity by 2-fold for both ftzQ (compare column 5 vs. 4, Fig. 2-13) and VP1
(column 5 vs. 4, Fig. 2-14), and 3-fold for LpHSF8 (column 5 vs. 4, Fig. 2-15A).
Consistent with the results obtained with TBP, with TBPm3 the E146R mutation also
showed more severe inhibition than E144R, with reductions in activity ranging from 6-
fold for ftzQ (compare column 6 vs. 4 and 5, Fig. 2-13) to 20-fold for LpHSF8 (column
6 vs. 4 and 5, Fig. 2-15A). The double mutation E144R/E146R, again, showed the
strongest inhibition for all the three activation domains, exhibiting up to a 55-fold
reduction in activity for LpHSF8 (compare column 7 vs. 4, Fig. 2-15A). Inhibition of


GUS/LUC
66
AtTBP2
AtTBP2
Figure 2-12. The effects of TBP concentration on transcriptional activation
in maize cells. Different amounts of T7-AtTBP2 DNA were coexpressed
with either CaMV 35S/GUS reporter whose activity is endogenous to maize
cells, or the GAL4/GUS reporter activated by Gal4 DBD/ftzQ. Total
amounts of DNAs were kept constant by addition of the T7 vector
expressing only the T7-epitope. GUS activity was normalized by the internal
control LUC activity and expressed as arbitrary units of relative nM Mu/cpm.
The results of one representative experiment are shown. The data for each
treatment was the average of three replicates.


53
when necessary). Binding reactions were for 1 hr at 4C in 300 pi of lx protein binding
buffer containing 0.1% BSA in a continuously rotated tube. The beads were then
extensively washed with lx protein binding buffer (4 times, 1 ml each). The bound
protein molecules were finally resolved by SDS-PAGE and detected with an anti-T7
monoclonal antibody (Novagen) used in conjunction with the ECL system (Pharmacia).
Binding efficiency was quantified by an imagine analysis program (Scionimagin).
Results
The C-terminal Stirrup of Arabidopsis TBP is Required for Binding to TFIIB
To test the role of the C-terminal stirrup domain of plant TBP in binding TFIIB,
single or double substitution mutations E144R, E146R and E144R/E146R of AtTBP2
were constructed and then evaluated in binding studies with AtTFIIB in vitro. Protein-
protein interactions in the absence of DNA (TATAA element) were determined using
GST pull-down assays. Binding was quantified using an image analysis program
(Scionimage) with images scanned from X-ray film. In Fig. 2-4A, free TFIIB is shown
to bind immobilized GST-TBP and its mutants, indicating that TBP/TFIIB interaction
does not require pre-association of TBP with the TATAA motif. The binding efficiency
of TBP was significantly reduced by the C-terminal stirrup mutations (Fig. 2-4A). The
two single mutations, E144R and E146R, exhibited an approximately 50% reduction in
binding, while the double mutation E144R/E146R showed more than an 8-fold
reduction (Fig. 2-4B). During in vitro translation, synthesis of a C-terminal deletion
mutant of TFIIB was also evident, as indicated by the faster migrating band in Fig. 2-
4A. Although the amount of input protein for the truncated TFIIB was almost equal to
that of the wild type protein, this deletion mutant bound to TBP and its mutants with


GUS/LUC
78
GAL4-dependent transcription
1200 -
900 -
T7
vector
600
300
0
ftzO-Ga!4 AD
VP16-Gal4 AD
TBP
T7
vector
E146R
E144R
E146R
4
TBP
L
6
E146R
JL
E144R
E146R
I
8
Effector constructs coexpressed with either ftzQ-
Gal4 AD or VP16-Gal4 AD double activators
Figure 2-18. Double activators show variable dependence on TBP-TFIIB
interactions. Gal4 DBD/ftzQ-Gal4 AD (left panel) or Gal4 DBD/VP16-Gal4 AD
(right panel) double activators were coexpressed in transient assays with the
GAL4/GUS reporter and wild-type AtTBP2 or the stirrup mutants. T7 vector
represents the empty vector control utilizing endogenous TBP. The results of one
representative experiment with three replicates are shown.


129
Horikoshi, M., Wang, C. K., Fujii, H., Cromlish, J. A., Weil, P. A., and Roeder. R. G.
(1989). Cloning and structure of a yeast gene encoding a general transcription
initiation factor TFIID that binds to the TATA box. Nature 341, 299-303.
Horikoshi, M., Yamamoto, T., Ohkuma, Y., Weil, P. A., and Roeder, R. G. (1990).
Analysis of structure-function relationships of yeast TATA box binding factor
TFIID. Cell 61, 1171-1178.
Ichimura, T., Isobe, T., Okuyama, T., Yamauchi, T., and Fujisawa, H. (1987). Brain 14-
3-3 protein is an activator protein that activates tryptophan 5-monooxygenase
and tyrosine 3-monooxygenase in the presence of Ca2+, calmodulin-dependent
protein kinase II. FEBS Lett. 219, 79-82.
Ichimura, T., Ito, M., Itagaki, C., Takahashi, M., Horigome, T., Omata, S., Ohno, S., and
Isobe, T. (1997). The 14-3-3 protein binds its target proteins with a common site
located towards the C-terminus. FEBS Lett. 413, 273-276.
Ichimura, T., Uchiyama, J., Kunihiro, O., Ito, M., Horigome, T., Omata, S., Shinkai, F.,
Kaji, H.. and Isobe, T. (1995). Identification of the site of interaction of the 14-3-
3 protein with phosphorylated tryptophan hydroxylase. J. Biol. Chem. 270,
28515-28518.
Imbalzano, A. N., Kwon, H., Green, M. R., and Kingston, R. E. (1994a). Facilitated
binding of TATA-binding protein to nucleosomal DNA. Nature 370, 481-485.
Imbalzano, A. N., Zaret, K. S., and Kingston, R. E. (1994b). Transcription factor (TF)
IIB and TFIIA can independently increase the affinity of the TATA-binding
protein for DNA. J. Biol. Chem. 269, 8280-8286.
Ingham. P. W., Baker, N. E., and Martinez-Arias, A. (1988). Regulation of segment
polarity genes in the Drosophila blastoderm by fiishi tarazu and even skipped.
Nature 331, 73-75.
Ingles, C. J., Shales, M., Cress, W. D., Triezenberg, S. J., and Greenblatt, J. (1991).
Reduced binding of TFIID to transcriptionally compromised mutants of VP16.
Nature 351, 588-590.
Inostroza, J., Flores, O., and Reinberg, D. (1991). Factors involved in specific
transcription by mammalian RNA polymerase II. Purification and functional
analysis of general transcription factor HE. J. Biol. Chem. 266, 9304-8.
Irie, K., Gotoh, Y., Yashar, B. M., Errede, B., Nishida, E., and Matsumoto (1994).
Stimulatory effects of yeast and mammalian 14-3-3 proteins on the Raf protein
kinase. Science 265, 1716-1719.


45
stirrup of yTBP in order to eliminate potential stabilizing effects between an activator
and components of TFIID or the holoenzyme. Stirrup mutations E186A, El88A and
LI 89 A in LexA-yTBP were all able to support high levels of transcription indicating
that a functional yeast PIC can be assembled without involvement of an activation
domain, even when the TBP-TFIIB interaction is disrupted by mutation (Lee and Struhl,
1997). Moreover, both E188A and LI89A mutations supported transcription dependent
on LexA-Gcn4 and LexA-yTFIIB as efficiently as the wild type yTBP (Lee and Struhl,
1997). In a separate study, yTFIIB mutants were created that were incapable of binding
to yTBP and VP 16 (Chou and Struhl, 1997). These TFIIB mutants supported
transcription by a series of different activation domains with both synthetic and natural
promoters as efficiently as wild type TFIIB. Paradoxically, a yTFIIB mutation
enhancing TATA-TBP-TFIIB ternary complex formation failed to support basal and
activated transcription in vitro; however, this mutation did support activated
transcription in vivo at lower levels (Bangur et al., 1997). These results strongly argue
that the TBP-TFIIB interaction may be either redundant to other interaction pathways, or
nonessential, in the process of PIC assembly in yeast cells. Overall, it is apparent that
the TBP-TFIIB interaction in yeast is not as critical as in humans with respect to
supporting PIC assembly and transcription.
In plants, although both TBP and TFIIB cDNAs have been isolated, few studies
have examined function or addressed questions related to the roles of protein-protein
interactions in the process of activated transcription. In this chapter, the functional
importance of the TBP-TFIIB interaction is characterized in both basal and activated
transcription by transient expression assays in maize cells. Maize suspension cells and


ACKNOWLEDGEMENTS
I am deeply grateful to my mentor, Dr. Bill Gurley, for his valuable professional
advice and personal support, which have made this work and my future career possible.
I also thank Dr. Rob Ferl for his collaboration in this study and valuable advice as a
committee member. Thanks are extended to Drs. Ken Cline, Don McCarty, and James
Preston III for their expertise, lab resources, and valuable advice as committee members;
to Drs. Curt Hannah and Charles Guy for their lab resources; to Dr. Prem Chourey for
the maize cell line; to present and past members of the Gurley lab: Drs. Eva Czamecka-
Vemer, Don Baldwin and Eloise Adams for their valuable clones and discussion; to the
DNA Sequencing Core of Microbiology and Cell Science Department, University of
Florida, for confirming the mutation constructs; to the students, staff, and faculty of
PMCB Program and Microbiology and Cell Science Department for their friendship.
With deep gratitude I thank my family for their support and understanding throughout
the course of this study.


25
and Struhl, 1997). Correspondingly, the TBP interaction mutants of TFIIB show no
impairment in supporting transcription by several activators (Chou and Struhl, 1997).
Interestingly, one yTFIIB point-mutation, R64E, significantly enhances formation of the
TATA-TBP-TFIIB complex, yet greatly inhibits both the basal and activated
transcription (Bangur et al., 1997). Although some of these mutants affect yeast cell
viability somewhat, all of these results strongly suggest that the TBP-TFIIB interaction
is not generally critical for transcriptional activation in yeast cells. Therefore, the
association of holoenzyme and TFIID in yeast cells may be mediated in large part by
other types of interactions between the two complexes outside of the TBP-TFIIB
interaction.
Unlike in yeast, in human cells the TBP-TFIIB interaction appears to be vitally
important for activated transcription. The same C-terminal stirrup mutations of hTBP
abolished basal (Bryant et al., 1996) and activated transcription in both HeLa and COS
cells (Bryant et al., 1996; Tansey and Herr, 1997). In addition, the inhibitory action of
these TBP mutants on transcription activated by VP 16 and CTF activation domains is
suppressed by the reciprocal mutation, R169E, of hTFIIB (Tansey and Herr, 1997). In
this case, the charge-charge interaction between TBP and TFIIB was presumably
restored by the R169E mutation of hTFIIB. In contrast to the findings from yeast
systems, these results suggest that in human cells, the TBP-TFIIB interaction performs a
critical role in supporting PIC assembly and activated transcription. However,
transcription driven by Spl is not significantly affected by the same TBP mutations
(Tansey and Herr, 1997). It seems that the requirement for the TBP-TFIIB interaction is
not universal, since recruitment mechanism of the PIC components by Spl somehow


120
A common theme in the 14-3-3 literature is that the 14-3-3 proteins function
primarily in the dimer state. This conclusion is supported by evidence that these proteins
readily form homo- or heterodimers in vitro and in vivo (Jones et al., 1995; Luo et al.,
1995; Wu et al., 1997). Protein structure determinations for 14-3-3s have further
proposed that the internal "negative groove" formed by the dimer is responsible for
interactions with other proteins (Liu et al., 1995). However, recent studies, together with
findings in this study, have challenged this model by showing that dimer structure, and
hence the negative groove, is not generally required for all interactions, since the
isolated box-1 domain, or isolated helix-7, is sufficient for binding to Raf-1 (Ichimura et
al., 1997), Bcr (Ichimura et al., 1997), TPHase (Ichimura et al., 1995), TBP, and TFIIB.
However, the possibility that the dimerization of 14-3-3s may have a regulatory role in
facilitating or inhibiting interactions with partner proteins can not be excluded.


8
In addition to co-activator roles, some TAFs also have affinity for DNA and
enzymatic activity as well. For the TATA-less core promoter of class II genes, the TBP
protein is unable to specifically bind to DNA. This problem can be overcome by the
initiator element which is located around the transcription start site. This element can be
recognized by some TAF proteins that seem to tether the TFIID complex at the core
promoter allowing for transcription (Martinez et al., 1994). Interestingly, some TAFs
appear to have enzymatic functions as seen with TAF250 which has both kinase and
acetyltransferase activities (Dikstein et al., 1996; Mizzen et al., 1996). The protein alone
or in TFIID is able to phosphorylate the RAP74 subunit of TFIIF at its serine residues
(Dikstein et al., 1996). The phosphorylation is done by the cooperative action of the two
kinase domains of TAF250, with each capable of autophosphorylation. It is thought that
this phosphorylation may provide a signal within the PIC for transcriptional regulation
(Dikstein et al., 1996). On the other hand, the histone acetyltransferase activity of
TAF.,250 (Mizzen et al., 1996) may be important in dissociating the histone core from
DNA to facilitate nucleosome remodeling, since the acetylation of histone proteins
inhibits histone-DNA interactions (Mizzen et al., 1996; Puig et al., 1998). Another
example is seen with human TAF170 which was shown to have an ATPase activity
(Chicca et al., 1998). hTAF170 binds to the TBP core region in the TBP-DNA complex
and hydrolyses ATP. The generated energy contributes to the dissociation of TBP from
DNA and TAF170 from TBP as well (Chicca et al., 1998). As expected, transcription is
inhibited by this ATPase activity when TBP is used in the reaction. However, either
TFIID or TFIIA included in the transcriptional reaction appears to be able to reverse the
TAFn170-mediated repression (Chicca et al., 1998).


54
A. Immobilized GST-TBP; TFIIB free
T7-ATFIIB
T7-AtTFIIBAC
WT E144R E146R E144R
E146R
GST-AtTBP2
10% input
B.
WT
AC
Bound
r
1
u

1
_
WT
E144R
E146R E144R
E146R
GST-AtTBP2
Figure 2-4. Binding of free TFIIB to immobilized GST-TBP2 or its mutants.
(A) Western blot of bound TFIIB. T7-AtTFIIB was synthesized by a coupled
transcription/translation reaction in wheat germ. T7-AtTFIIBAC represents
prematurely terminated protein resulting in an undefined C-terminal truncation.
Bands were visualized by probing the western blot with anti-T7 monoclonal
antibody. (B) Quantification of imagines for (A). Bands on X-ray film were
scanned and then analyzed by computer using Scionimage software. Area units
are arbitrary.


34
al., 1993). The AD still retains lull transcriptional activity when the acidic amino acids
are replaced by a cluster of positive residues (Leuther et al., 1993). In yeast cells, over
expression of GAL4 protein squelched transcription of the genes without the GAL4 site,
a phenomenon believed to be due to the titration of the general transcription factors)
(Gill and Ptashne, 1988). Indeed, the GAL4 AD is able to interact with both TBP and
TFIIB, and transcription likely is mediated by these interactions in vivo (Wu et al.,
1996).
VP16
VP 16 is a human herpes simplex virus (HSV) protein that is the transcriptional
activator for the five immediate early genes of the virus. The protein itself does not have
affinity for promoter DNA, instead, it relies on other transcriptional factors of the human
host cells (Triezenberg et al., 1988a). Promoter binding activity is provided by the host
protein Oct-1, which recognizes the 5-TAATGARAT-3 sequence of the viral promoter
(Stem et al., 1989). However, the binding of Oct-1 to the promoter does not lead to
transcriptional activation (Triezenberg et al., 1988b). Transcription can not occur until
VP 16 joins to Oct-1 with the assistance of other proteins called host cellular factors
(HCF) to form the multiprotein-DNA complex (Lai and Herr, 1997). The
phosphorylation of S375 of VP16 outside the activation domain by CKII is required for
the assembly of the activator complex in vitro and further the transactivation by the
complex in vivo (OReilly et al., 1997).
The strong acidic activation domain of VP 16 is located at the end of the C-
terminus of the protein from aa 413-490 (Cousens et al., 1989). The activation domain
can be further divided into two subdomains: one at aa 413-456 and the other from aa


108
14-3-3 <(), TBP2 and TFIIB were all from arabidopsis. Unlike mutation by deletion, the
relatively mild mutation by alanine substitution was assumed to have a minimal effect
on the overall structure of the 14-3-3 protein. Helix-7 of 14-3-3 was targeted for
mutation due to its strong evolutionary conservation, being identical between many 14-
3-3 isoforms of different origins including AU4-3-3 , hl4-3-3 u and hl4-3-3 t|. In the
context of full-length 14-3-3, every two residues of helix-7 in pair (aa 177-
PIRLGLALNFSVFYYEI-193) were systematically substituted with two alanines in
eight constructs to scan the entire helix. As indicated by lanes 5, 6, and 7 in Fig. 3-7, a
clear footprint in 14-3-3 binding activity was obtained for both At TFIIB and AtTBP2.
The F186A/S187A mutation nearly eliminated interactions with AtTFIIB and AtTBP2
(compare lane 6 vs. 1, Fig. 3-7), indicating that these two residues are critically
important for the interaction. Flanking mutations (L184A/N185A and V188A/F189A)
showed partial loss of binding capacity for AtTFIIB and AtTBP2 (compare lane 5 vs. 1,
and 7 vs. 1, Fig. 3-7). Other residues more distal to FI86 and SI87 appeared to have no
involvement in interactions with AtTBP2 or AtTFIIB, since corresponding mutant
proteins still showed strong bindings (compare lanes 2, 3, 4, and 8 vs. 1, Fig. 3-7). The
contributions of residues Y190 and Y191 were not tested. As a whole, these results
clearly show that the in vitro interactions between At 14-3-3 <}> and either AtTBP2 or
AtTFIIB were highly specific and required participation of either one or both of the
residues FI 86 and SI 87 of helix-7.
At 14-3-3 <(> Stimulates GAL4/GUS Expression in Onion Cells
The transcriptional potential of 14-3-3 proteins was assessed in plant cells by
monitoring the activity of a Gal4 DNA binding domain/14-3-3 fusion protein in


90
conserved because the negative residues are conserved between the numerous isoforms.
This negative groove is thought to be a site for protein-protein interactions (Liu et al.,
1995). In recent studies, 14-3-3s were designated as phosphoserine-binding proteins
because many of their interacting proteins contain the consensus motif RSXpSXP and
require a phosphorylated serine residue for interactions to occur (Muslin et al., 1996).
The binding of a 14-3-3 protein results in inhibition of dephosphorylation of the
phosphoserine in the partner protein (Bachmann et al., 1996; Banik et al., 1997).
Phosphoserine binding was thought to serve as a general mechanism for 14-3-3 proteins
to participate in a variety of diverse cellular functions (Muslin et al., 1996). Domain
mapping for 14-3-3 proteins has identified a C-terminal region called box-1 which is
able to independently mediate phosphoserine binding (Ichimura et al., 1997). The
isolated box-1 domain can not form a dimer structure, yet still retains full capacity for
binding other proteins (Ichimura et al., 1997; Ichimura et al., 1995). Therefore, the
importance of the dimeric structure and the role of the negative groove of 14-3-3
proteins in protein-protein interactions remain unclear.
Although phosphoserine binding can explain most interactions involving 14-3-3
proteins, there are several exceptions. For example, the Cl domain of myosin II heavy-
chain-specific protein kinase C (MHC-PKC), which was shown to interact with 14-3-3
protein, does not contain the phosphoserine motif (Matto-Yelin et al., 1997). In the case
of the glucocorticoid receptor, the potential RSXpSXP motif is present only in its
activation domain, not in the ligand binding domain where 14-3-3 r| preferentially binds
(Wakui et al., 1997). These exceptions suggest that phosphoserine binding is but one of
several possible modes of interactions with 14-3-3 proteins.


14
activated transcription (Hansen et al., 1997). This finding suggests that a homo log of
TBP is required for the cell-type-dependent transcription in addition to its general roles
in transcription.
The protein structure of the arabidopsis TBP iso form 2 has been revealed by X-
ray crystallography studies (Kim and Burley, 1994; Nikolov and Burley, 1994). AtTBP2
is the smallest TBP protein known, and contains only 200 amino acid residues. The total
molecule is almost just the core domain with an N-terminal loop of only eighteen amino
acids (Gasch et al., 1990). In protein crystals, AtTBP2 is organized as two structural
repeats (Kim and Burley, 1994; Nikolov and Burley, 1994). The structures of the two
repeats are very similar and show strong twofold symmetry. Each repeat contains two a-
helices (almost perpendicular to each other), five anti-parallel p-sheets and one stirrup
like loop between the second and third P-sheets. The ten P-sheets in total form a
concave surface of the TBP molecule and are responsible for binding to DNA. The
upper convex surface of the molecule is composed of the four a-helices, the basic linker
between the two repeats and the N-terminal loop (Kim and Burley, 1994; Nikolov and
Burley, 1994). This surface is not in contact with DNA and is presumably responsible
for interactions with TAFs, holoenzyme components and other transcriptional regulator
proteins.
Interaction with the TATA element of the core promoter is the primary function
of TBP in transcription of class II genes and has been characterized in great detail. The
TBP protein is believed to exist as two forms: monomer and dimer (Coleman et al.,
1995; Taggart and Pugh, 1996). The dimer does not have DNA binding activity and has
to dissociate into the monomer form for binding to DNA (Coleman and Pugh, 1997).


no
transient assays. Fusion of the Gal4 DBD to the N-terminus provided a means of
tethering the 14-3-3 protein to the promoter in addition to providing a nuclear
localization signal. Transcriptional activity was monitored using a GUS reporter driven
by a minimal CaMV 35S promoter containing multiple copies of the Gal4 DNA binding
site upstream. A second reporter comprised of the luciferase gene driven by a maize
ubiquitin promoter (Christensen and Quail, 1996) was used as an internal control for
transformation efficiency. The reporter and effector constructs used in this study are
diagramed as in Fig. 3-8, and a representative experiment is shown in Fig. 3-9. In
several repeated experiments using onion epithelial cells, coexpression of Gal4 DBD/14-
3-3 using a low amount of DNA (2 pg) showed no significant stimulation of GUS
expression compared to Gal4 DBD alone. However, when high amounts of DNA (20
pg) were used, activity was stimulated approximately 5-fold over background levels.
Although the GAL4/14-3-3 protein is transcriptionally active in onion cells, the level of
stimulation was low compared to a strong activator such as the Gal4 activation domain.
To address the possibility that the low activation potential of the tethered 14-3-3
protein was related to the cell type used for transient assays, the same experiments were
repeated in maize Black Mexican Sweet suspension cells. Surprisingly, no Gal4
DBD/14-3-3-dependent activity was seen (Fig. 3-10A). To resolve this paradox,
experiments were conducted to confirm that Gal4 DBD/14-3-3 protein was expressed in
maize cells and was functional in DNA binding. Due to the low amounts of protein
expressed in transient assays, GAL4/14-3-3 levels were evaluated by an indirect assay
based on suppression of a strong activator fused to the Gal4 DBD. If the GAL4/14-3-3
protein is stable and has no transcriptional activity, coexpression with the highly active


114
Drosophila ftzQ activator (GAL4/ftzQ) would result in an inhibition of GAL4/ftzQ
activity. The Drosophila ftzQ activation domain showed strong activation of
GAL4/GUS expression when fused with the Gal4 DBD (Fig. 3-10A). As expected,
coexpression of Gal4 DBD alone with Gal4 DBD/ftzQ greatly suppressed GUS
expression; however, coexpression of Gal4 DBD/14-3-3 with Gal4 DBD/ftzQ resulted
in no or much less suppression of ftzQ activity (Fig. 3-1 OB). Note that 2 pg of Gal4
DBD/ftzQ DNA was used when coexpressed with Gal4 DBD or Gal4 DBD/14-3-3. It is
assumed that activity of 2 pg Gal4 DBD/ftzQ DNA is roughly equal to 50% of that
obtained using 4 pg of DNA. The simplest interpretation of these results is that unlike
Gal4 DBD, the expressed levels of Gal4 DBD/14-3-3 were too low to effectively
compete for DNA binding against Gal4 DBD/ftzQ. This finding, together with the
observation that high levels of DNA were needed to detect transcriptional activity in
onion cells, suggests that the low or no activity shown by Gal4 DBD/14-3-3 was due to
low expression levels of functional protein.
Discussion
Using in vitro GST pull-down assays, direct physical interactions of plant and
human 14-3-3 proteins with TBP and TFIIB were observed. Additionally, human 14-3-3
interacted with hTAF32, but not hTAFu55. These results provide evidence that the 14-
3-3 proteins, when associated with a DNA binding complex or DBD, may directly
participate in the transcriptional activation of genes through contacts with the PIC.
Several studies have shown that 14-3-3s associate with a transcriptional regulatory
complex through interactions with transcriptional activators such as VP1 (Schultz et al.,
1998), EmBPl (Schultz et al., 1998), or the glucocorticoid receptor (Wakui et al., 1997).


86
compared to recruitment dependent on a single type of activation domain and lessen the
importance of any single interactions, including TBP-TFIIB contact. Impairment of one
interaction pathway, such as TBP-TFIIB contact, may be compensated by alternate
pathways present in complex natural promoters.
Consistent with this view, activator constructs containing the two activation
domains VP 16 and the Gal4 AD were less affected by TBP stirrup mutants than the
corresponding single activation domains, showing only 45% inhibition with the double
mutation E144R/E146R (Fig. 2-18). However, this lessened dependence on the TBP-
TFIIB interaction by multiple activation motifs is not universal, since the ftzQ-Gal4 AD
double activator was still very sensitive to TBP stirrup mutations, with activity reduced
5-fold by the E144R/E146R mutation. Although it has been demonstrated that an
artificial activator comprised of two different activation motifs may show a reduced
requirement for the TBP-TFIIB interaction, unexpectedly, this dependence is also
specific to the particular combination of activation motifs employed.
An important assumption underlines much of the interpretation of results
presented in this study that the mutations introduced into the C-terminal stirrup of
AtTBP2 do not affect stability of the protein. Since the transiently expressed proteins
were not synthesized in sufficient amounts to be detectable by western blotting using
chemiluminescent visualization, the question remains. However, there are several lines
of indirect evidences supporting the assumption that the mutations did not affect TBP
levels in maize cells. First, basal transcription was stimulated by both wild type and
mutant TBP at similar levels (Fig. 2-6). Second, similar levels of stimulation between
wild type and mutant TBPs were observed for the CaMV 35S promoter (Fig. 2-16).


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12
polymerase is believed to enable the enzyme to overcome transcriptional repression by
chromosome structure.
The SRB proteins of the mediator complex appear to play both positive and
negative roles in transcription. With temperature-sensitive mutations in the SRB genes,
yeast cells show a rapid and general shutdown of mRNA synthesis when transferred to
the restrictive temperature, suggesting that some SRBs are required for transcriptional
activation (Thompson and Young, 1995). The positive effects by the SRBs may be
related to their roles in serving as the targets for activator proteins. The study by Koh et
al. (1998) has clearly shown that in vivo Srb4 enhances transcription by Gal4 AD
through direct interaction between the two proteins, since reciprocal mutations in both
proteins can restore transcriptional activity. Srb4 may serve as a general target for many
types of activation domains, because it is an essential factor for expression of most class
II genes in yeast (Lee et al., 1998). On the other hand, several other Srbs exert a
negative influence on transcription. The human NAT complex contains Srb7, SrblO and
Srbl L and is a negative regulator of activated transcription (Sun et al., 1998). It
associates with the human RNA polymerase II holoenzyme and phosphorylates the CTD
domain at residues distinguished from those by the kinase of TFIIH (Sun et al., 1998).
However, it is unknown how this activity can negatively regulate transcription. In some
cases, the negative SRBs can function by serving as the targets for the repressors. For
example, the yeast repressor Sfll represses the SUC2 gene by recognizing the repression
site located immediately 5 to the TATA element and directly targeting Srb 9 and Srbl 1
(Song and Carlson, 1998). Presumably, the interactions of Sfll with these SRB proteins


LIST OF REFERENCES 121
BIOGRAPHICAL SKETCH 143
VI


GUS/LUC
60
Transcription activated by Gal4 DBD/ftzQ
TBP mutants
1 2 ; 3 4 5 6
Effector constructs coexpressed with Gal4 DBD/ftzQ
Figure 2-7. Effects of coexpression of TBP or the C-terminal stirrup
mutants on ftzQ transcriptional activity. Transcription of the GUS gene
was activated by Gal4 DBD/ftzQ bound to the GAL4 sites (lOx)
upstream of the CaMV 35S minimal promoter (-46). The same amount
of DNA (10 pg) was used for co-expression of each effector construct.
Transcriptional activity was normalized by the internal control LUC
activity and expressed as arbitrary units of relative GUS/LUC (nM
Mu/cpm). The results of one representative experiment are shown. The
data for each treatment was the average of three replicates.


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7
characterized by in vitro biochemical approaches. The importance of each individual
interaction for TFIID function in living cells remains to be demonstrated.
Functional analysis of the TFIID complex reveals distinct requirements for
TAFs in both basal and activated transcription. In the Drosophila in vitro transcription
system, the TBP/TAF250 binary complex is sufficient to support the basal transcription,
but not activated transcription, which requires additional TAF proteins depending on
the specific transactivator bound to the promoter (Chen et al., 1994). For example,
dTAF150 and dTAFl 10 are required for transcription activated by NTF-1 and Spl,
respectively (Chen et al., 1994). In these systems, dTAFs are believed to play specific
co-activator roles (Chen et al., 1994). The importance of TAFs in activated
transcription is further confirmed in vivo where dTAFs 60 and 100 mutants failed to
support transcription of bicoid-dependent target genes during Drosophila embryo
development (Sauer et al., 1996). In contrast to a clear dependence on TAFns for
activated transcription in metazoans, deletion or inactivation of several TAF genes in
yeast does not affect transcription by several activator proteins, indicating that TAFs are
not generally required in activated transcription in yeast (Moqtaderi et al., 1996; Walker
et al., 1996). Although, apparently, there is a functional TFIID complex in yeast cells
(Gonzalez-Couto et al., 1997), it is likely that not all TAFs are generally required in
activated transcription, but, rather, they are selectively used in an activator- or promoter-
dependent manner. Even in animals, the co-activator function of TAFus seems to be
redundant with other general co-activator proteins like PC4 (positive co-activator 4) and
pX which are able to support transcription in the absence of TAFs in the human in vitro
transcription systems (Haviv et al., 1996; Wu et al., 1998).


106
A.
5% 5%
input 1 2 input 3 4
T7-hTBP
hTFIIB-T7
14-3-3 14-3-3
B.
5% 5%
input 1 2 input 3
GST- GST GST-
14-3-3 14-3-3
3SS-hTAFu55
3SS-hTAF32
Figure 3-5. Immobilized hi4-3-3 u interacted with human TBP, TFIIB and
TAF32, but not with TAF55. GST, GST-14-3-3, T7-hTBP and T7-hTFIIB
proteins were expressed in E. coli. Human TAF32 and hTAFn55 proteins
were in vitro translated using the rabbit reticulocyte TNT system (Promega)
and labeled by 35S-methionine. Binding reactions were conducted at 4C for 3
hr in buffer containing 0.15 M KC1. (A) Binding reactions between
immobilized hl4-3-3 o and human TBP and TFIIB. Bound T7-hTFIIB
andT7-hTBP were detected on western blots using anti-T7 antibody. (B)
Binding reactions between immobilized hi4-3-3 u and 35S-labeled hTAFn32
(lane2) and hTAF55 (lane 3). 35S-labeled proteins were detected by PPO-
enhanced autoradiography.


138
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CHAPTER 2
CRITICAL ROLE OF THE TBP-TFIIB INTERACTION IN
SUPPORTING ACTIVATED TRANSCRIPTION IN PLANT CELLS
Introduction
Transcription of class II genes in eukaryotic cells is directed by the interaction
between two megacomplexes of proteins, TFIID and RNA polymerase II holoenzyme.
TFIID provides promoter-binding activity, and the holoenzyme contains the catalytic
function for mRNA synthesis. The interaction of these two complexes results in
formation of the PIC, the final configuration of factors before the initiation of
transcription. Therefore, mechanisms that facilitate the recruitment of TFIID and
holoenzyme to the promoter enhance formation of the PIC and lead to gene activation.
Although activator proteins usually function by binding to the promoter and then to
general transcription factors to recruit them to the promoter, general transcription factors
can also be directly tethered to the promoter by translational fusions with heterologous
DNA binding domains (DBDs). In yeast cells, the pure recruitment of either TFIID or
holoenzyme to the promoter by directly tethering TAFs, or TFIIB can lead to high
levels of transcription comparable to that achieved by a strong activator such as VP 16
(Gonzalez-Couto et al., 1997). In this tethered system, recruitment of the first complex
to the promoter presumably results in the subsequent recruitment of the second complex.
Apparently, recruitment of the second complex is mediated by interactions between
components of TFIID and the holoenzyme. Biochemical studies in vitro have indicated
39


125
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I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
William B. Gur
Associate Professor of
Microbiology and Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Phi
Kenneth C. Cline
Professor of Horticultural Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fulfy-ttdeqtiqii, in scope and
quality, as a dissertation for the degree of Doctor ofPnilosophy,
Ferl
rofessor of Horticultural Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Donald R.
Professor of Ho:
cience
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
fames F. Preston III
Professor of
Microbiology and Cell Science


19
Phosphorylation of the CTD by a kinase activity of TFIIH is believed to disrupt the
TBP-CTD interaction and to release the polymerase from the promoter to proceed in the
synthesis of RNA (Parada and Roeder, 1996). The functional importance of this step in
transcription has been confirmed by an in vivo study in Drosophila embryonic cells
(Yankulov et al., 1996).
General Transcription Factor TFIIB
TFIIB is another highly conserved general transcription factor among eukaryotic
organisms. The protein in general consists of two domains: the N-terminal domain and
the C-terminal core. The N-terminal domain is composed of about 100 aa residues and
contains the zinc-ribbon and the adjacent highly conserved region. The C-terminal core
is composed of more than 200 aa, and contains two imperfect direct repeats linked by a
small region characteristic of basic amino acids. The structure of the C-terminal core,
instead of the full-length hTFIIB, has been characterized by both crystallography
(Nikolov et al., 1995) and NMR in solution (Bagby et al., 1995), because inclusion of
the N-terminal region resulted in structural instability of the protein. The core domain is
a twofold symmetrical molecule as reflected in the positions of two imperfect repeats.
Each repeat consists of five a-helices with an additional helix at the C-terminal end of
the second repeat (Bagby et al., 1995; Nikolov et al., 1995). The a-helices of the first
repeat are designated as Al to El, and their counterparts in the second repeat as A2 to
E2 (Bagby et al., 1995). The El helix is amphipathic with respect to the distribution of
positive residues, and is thought to be a critical domain involved in interactions with
TBP, DNA (Nikolov et al., 1995) and activators such as VP 16 (Roberts et al., 1993).
Protease topology analysis on full-length hTFIIB indicates that the N-terminal domain


29
TFIIB strongly support models for activated transcription that incorporate recruitment of
general transcription factors through protein-protein interactions between activator
proteins bound to the promoter and their target proteins within the PIC, and specifically
support conclusions that TBP and TFIIB can serve as crucial targets for activation
domains.
Despite good evidence for the recruitment model for activated transcription, it is
important to note that a potential for interaction demonstrated in vitro between an
activator protein, or motif, and a general transcription factor does not necessarily imply
that this potential for interaction will actually be important, or crucial in activating
transcription in a living cell. For example, mutations of either TBP or TFIIB defective of
interacting with VP 16 had no effect on transcription activated by VP 16 in vivo (Chou
and Struhl, 1997; Tansey and Herr, 1995). These results suggest that transcriptional
activation by VP 16 may be mediated by recruitment pathway(s) not involving either
TBP or TFIIB. This apparently paradoxical finding may be due to the ability of VP 16 to
bind to multiple PIC members like TAFs (Goodrich et al., 1993), TFIIH (Xiao et al.,
1994), TFIIA (Kobayashi et al., 1995) and co-activator PC4 (Ge and Roeder, 1994) in
addition to TBP and TFIIB, as shown by in vitro biochemical studies. Given such
possible multiple interactions in vivo, one would not expect a dramatic affect on
transcription by the disruption of a single interaction because of the redundant
recruitment pathways. Theoretically, TFIID and holoenzyme can be readily recruited by
an interaction with any one of the components.
In some cases, a direct interaction between TBP or TFIIB and a particular
activation domain is not sufficient for transcriptional activation. Activator proteins in


44
1996). Similar studies done in HeLa cells showed that E284R and E286R totally
abolished activated transcription by CTF or VP16, but mutation L287A resulted in much
less inhibition (Tansey and Herr, 1997). Using VP 16 activity for comparison, the
importance of each individual residue in the C-terminal stirrup of hTBP in activated
transcription appears to be cell-type-dependent, although all are required for in vitro
binding in EMSA assays. To further support the conclusion that transcriptional
impairment caused by stirrup mutations is due to the disruption of the TBP-TFIIB
interaction, the reciprocal mutation in hTFIIB, R169E, was shown to specifically
suppress the inhibitory effects of the E284R mutation, presumably by restoring the
charge-charge interaction between TBP and TFIIB (Tansey and Herr, 1997).
Not only are there differences in the relative importance of individual amino
acids (HeLa vs. COS cells), dependence on the TBP-TFIIB interaction for activated
transcription may also vary between different activators. For example, the Spl activator
is able to tolerant C-terminal stirrup mutations with showing only 50% reduction in
transcription when assayed in HeLa cells (Tansey and Herr, 1997), whereas VP 16, CTF
and p53 are highly sensitive to the same mutations. Differences in dependence on the
TBP-TFIIB interaction suggest that some activators may use different interaction
pathways for PIC assembly.
Surprisingly, the results obtained by yeast genetic approaches are contrary to
those obtained in human transient expression studies. When yTBP was fused to the
LexA DBD, high levels of transcription were achieved in the absence of an activator
protein due to the direct tethering of TBP to the promoter. This system, which
circumvents the need for activators, was used to evaluate mutations in the C-terminal


109
Helix-7 domain of 14-3-3
177--P 1RLGLALNFSVFYYE 1193
AA
AA
AA
AA
AA
AA
AA
P177A R179A G181A L184A F186A V188A E192A
WT I178A L180A L182A N185A S187A F189A I193A
5% input of
T7-14-3-3
binding to
GST-AtTFIIB
binding to
GST-AtTBP2
Figure 3-7. Analysis of alanine substitution mutations within the helix-7 domain
of 14-3-3 protein. In the context of full-length Atl4-3-3 , every two amino acid
residues of helix-7 were substituted with two alanines using the Altered Sites II-
Exl in vitro Mutagenesis System (Promega). These T7-tagged free mutant
proteins were released from GST by thrombin digestion and incubated at room
temperature for 2 hr with equal amounts of immobilized GST-AtTFIIB or GST-
AtTBP2 in binding buffer containing 0.15 M KC1. Bound proteins were detected
in western blots by anti-T7 antibody. A clear footprint of binding disruption is
seen in lanes 5 to 7 with residues FI86 and SI87 being the most critical (lane 6).


GUS/LUC
63
Transcription activated by Gal4 DBD/LpHSF8
TBP mutants
Effector constructs coexpressed with Gal4 DBD/LpHSF8
Figure 2-10. Effects of coexpression of TBP or the C-terminal
stirrup mutants on LpHSF8 AD transcriptional activity. The details
are the same as in Fig. 2-7.


91
The 14-3-3 proteins are also found as part of a transcriptional DNA binding
complex. In several plant species, including arabidopsis, rice and maize, 14-3-3 proteins
were reported to associate with G-box DNA binding complexes in vitro (Lu et al., 1992;
Schultz et al., 1998; de Vetten et al., 1992). In agreement with these results, 14-3-3
proteins were found in the nuclei of both maize and arabidopsis cells by confocal
microscopy (Bihn et al., 1997). In another study, human 14-3-3 r| was shown to
associate with the glucocorticoid receptor in a yeast two-hybrid screening. The
association with 14-3-3 q was through direct interaction with the ligand binding domain
(ligand bound state) of the glucocorticoid receptor. Over-expression of 14-3-3 r| in
COS-7 cells stimulates the glucocorticoid receptor-dependent transcription in a ligand
dependent manner (Wakui et al., 1997). These findings raise the possibility that 14-3-3
proteins may also participate in the regulation of transcription; however, the role for 14-
3-3 proteins in transcriptional regulatory complexes is not understood. In general, 14-3-
3s may either alter the iunction of activator proteins such as the glucocorticoid receptor
or the G-box binding factor, or they may be more directly involved in the recruitment of
general transcriptional factors within the pre-initiation complex.
Eukaryotic mRNA transcription requires the recruitment of the pre-initiation
complex (PIC) onto the promoter. This recruitment is usually accomplished through
protein-protein interactions between an activator and various members of the PIC
(Colgn et al., 1995; Colgn et al., 1993; Hadzic et al., 1995; Nakshatri et al., 1995;
Roberts et al., 1993). The activator can be a sequence-specific DNA binding protein
itself or a component of a multiprotein-DNA complex. In many cases, the TATA-box
binding protein (TBP) and the general transcription factor IIB (TFIIB) serve as


80
same interaction make only minor contributions to overall activity. Likewise,
interactions between TBP and TFIIB were not important in basal transcription from the
minimal CaMV 35S promoter. Although the same domains of TBP participate in
binding TFIIB in both human and plants, the relative contributions of individual residues
show clear distinctions between these organisms.
Using GST pull-down assays, direct protein-protein interaction between AtTBP2
and AtTFIIB was observed in vitro. The present experiments differ from many of the
previous studies by examining TBP-TFIIB interactions in the absence of TATAA
element DNA, which is commonly used in employing EMSAs (Chou and Struhl, 1997;
Lee and Struhl, 1997; Tang et al., 1996). This work, and a previous study using human
components (Ha et al, 1993), indicates that the association of TBP with DNA is not a
prerequisite for the association of TBP with TFIIB. In addition, the reduction in binding
of the inadvertent C-terminal deletion of TFIIB (Fig. 2-4) and the clear involvement of
the C-terminal stirrup of TBP in the TBP-TFIIB interaction are consistent with previous
studies in humans indicating that these domains are active in binding (Ha et al, 1993;
Hisatake et al., 1993).
Although the TBP-TFIIB interaction appears to also involve residues of the C-
terminal stirrup of TBP in plants, there are some significant differences between plants
and other organisms. In humans, TBP stirrup residues E284 and E286 (corresponding to
El44 and El46 in AtTBP2) are both critically important in binding with hTFIIB.
Mutations E284A and E286A showed more than 20-fold inhibition in binding (Tang et
al, 1996). Likewise, the same C-terminal stirrup mutations eliminated the binding for
yeast proteins in similar experiments (Lee and Struhl, 1997). Compared to human and


128
Hansen. S. K., Takada, S., Jacobson. R. H Lis. J. T., and Tjian. R. (1997).
Transcription properties of a cell type-specific TATA-binding protein. TRF. Cell
91, 71-83.
Haviv, I., Vaizel, D.. and Shaul, Y. (1996). PX. the HBV-encoded coactivator, interacts
with components of the transcription machinery and stimulates transcription in a
TAF-independent manner. EMBO J. 15, 3413-3420.
Heard. D. J., Kiss, T., and Filipowicz, W. (1993). Both Arabidopsis TATA binding
protein (TBP) isoforms are functionally identical in RNA polymerase II and III
transcription in plant cells: evidence for gene-specific changes in DNA binding
specificity of TBP. EMBO J. 12, 3519-3528.
Heller. H., and Bengal, E. (1998). TFIID (TBP) stabilizes the binding of MyoD to its
DNA site at the promoter and MyoD facilitates the association of TFIIB with the
preinitiation complex. Nucleic Acids Res. 26, 2112-2120.
Hill. A., Nantel, A., Rock. C. D., and Quatrano, R. S. (1996). A conserved domain of the
viviparous-1 gene product enhances the DNA binding activity of the bZIP
protein EmBP-1 and other transcription factors. J. Biol. Chem. 271, 3366-3374.
Hisatake, K., Ohta, T., Takada, R., Guermah, M., Horikoshi, M., Nakatani. Y., and
Roeder, R. G. (1995). Evolutionary conservation of human TATA-binding-
polypeptide-associated factors TAFII31 and TAFII80 and interactions of
TAFII80 with other TAFs and with general transcription factors. Proc. Natl.
Acad. Sci. USA 92, 8195-8199.
Hisatake, K., Roeder, R. G., and Horikoshi, M. (1993). Functional dissection of TFIIB
domains required for TFIIB-TFIID-promoter complex formation and basal
transcription activity. Nature 363, 744-747.
Hoecker, U., Vasil, I. K., and McCarty, D. R. (1995). Integrated control of seed
maturation and germination programs by activator and repressor functions of
Viviparous-1 of maize. Genes Dev. 9, 2459-2469.
Hoey, T., Dynlacht, B. D., Peterson, M. G., Pugh, B. F., and Tjian. R. (1990). Isolation
and characterization of the Drosophila gene encoding the TATA box binding
protein, TFIID. Cell 61, 1179-1186.
Hoffman. N. E., Ko, K., Milkowski, D., and Pichersky, E. (1991). Isolation and
characterization of tomato cDNA and genomic clones encoding the ubiquitin
gene ubi3. Plant Mol. Biol. 17, 1189-1201.
Holstege, F. C., van der Vliet, P. C., and Timmers, H. T. (1996). Opening of an RNA
polymerase II promoter occurs in two distinct steps and requires the basal
transcription factors IIE and IIH. EMBO J. 15, 1666-1677.


6
known TAFs proteins in animals and yeast. A second form of TFIID appears to be
tissue-specific, and is found associated with specific cell types in Drosophila (Hansen et
al., 1997). The tissue-specific TBP and TAFs in this complex are distinct from those
ubiquitous ones in the general TFIID and are named TRF and nTAFs respectively
(Hansen et al., 1997). Finally, there is another TFIID-like complex that lacks TBP
(Wieczorek et al., 1998). The complex contains several TAFs and other unknown
proteins. This TBP-less complex also has the normal function of TFIID in transcription
of both TATA-containing and TATA-less promoters in vitro, but the implication in
terms of physiological function is unclear.
For the general TFIID, its architecture appears to be highly conserved among
eukaryotic organisms. The largest TAF (hTAF,,250, dTAF250 and yTAF130) is
thought to serve as a primary anchor to TBP; the two proteins together act as a platform
for the entry of other TAFs into the TFIID complex by the interactions with either TBP
or the largest TAF (Burley and Roeder, 1996). For instance, in the assembly of
Drosophila TFIID, TBP interacts with TAFs 150 (Verrijzer et al., 1994), 80 (Kokubo et
al., 1993b), 62 (Kokubo et al., 1994; Weinzierl et al., 1993b), 42 (Kokubo et al., 1994)
and 30a/22 (Kokubo et al., 1994; Yokomori et al., 1993). dTAF250 interacts with
dTAFs 110 (Kokubo et al., 1993a; Weinzierl et al., 1993a), 150 (Verrijzer et al., 1994),
62 (Weinzierl et al., 1993b) and 30p (Yokomori et al., 1993). The small TAFs also
participate in interactions with each other to further stabilize the TFIID complex
(Kokubo et al., 1994; Kokubo et al., 1993b; Weinzierl et al., 1993b; Yokomori et al.,
1993). However, there is an important caveat in that these interactions are all


1780
1993
UNIVERSITY OF FLORIDA


49
Ubi/LUC
(Internal control)
+1
Ubiquitin promoter
Luciferase gene
Null
promoter/GUS
No promoter GUS gene
Minimal
promoter/GUS
35S/GUS
GAL4/GUS
+1
TATAA
CaMV 35S (-46)
GUS gene
+1
UAS TATAA
CaMV 35S promoter
GUS gene
+1
Gal4 (lOx) CaMV 35S
(-46)
GUS gene
Figure 2-2. Reporter plasmids: luciferase internal control and GUS test reporters.


GUS/LUC
74
A.
Transcription activated by wild-type
CaMV 35S promoter in maize cells
TBP mutants
150 n
E144R
120
90
TBP
60
T7
30 vector
E146R
E144R
E146R
E144R
E146R
K197E
Ill
3 4 5 6
Effector constructs coexpressed
with the CaMV 35S/GUS reporter
Figure 2-16. Effects of TBP and C-terminal stirrup mutants on
CaMV 35S activity in maize cells. (A) Different effectors with the
same amount of DNA were coexpressed with the CaMV 35S/GUS
reporter. CaMV 35S promoter activity in maize cells was expressed
as arbitrary units of relative GUS/LUC (nM Mu/cpm). The results of
one representative experiment with three replicates are shown.


116
previous reports that two bindings of 14-3-3 to glucocorticoid receptor and MHC-PKC
do not involve phosphoserine (Matto-Yelin et al., 1997; Wakui et al., 1997), our studies
make it apparent that 14-3-3 proteins participate in phosphoserine-independent
interactions.
The biological significance of these in vitro interactions is supported by the close
similarity between 14-3-3/TFIIB interactions and those of the mammalian viral activator
VP16 with TFIIB. Deletion analysis for hTFIIB revealed two 14-3-3 binding domains
localized to repeats 1 and 2 of the C-terminal conserved core, which appear to
correspond with the VP 16 binding domains previously characterized (Roberts et al.,
1993). Both 14-3-3 and VP 16 interact with the El helix of repeat 1 in a very similar
manner, since the R185E/R193E mutation within this helix disrupted binding for both
proteins. Furthermore, VP16/hTFIIB and 14-3-3/hTFIIB interactions are mutually
exclusive based on the competition between VP16 and 14-3-3 for binding to hTFIIB.
The finding that the 14-3-3/hTFIIB interaction are similar to the VP16/hTFIIB
interaction in vitro suggests that 14-3-3 proteins may be involved in the activation of
transcription in situations where they are components of DNA binding complexes.
The potential of 14-3-3 proteins to activate transcription was evaluated by using
the Gal4 DBD/14-3-3 chimera to activate GAL4/GUS reporter gene expression in vivo.
In onion epidermal cells, the 14-3-3 protein showed weak transcriptional activity, with
about 5-fold stimulation of GUS expression compared to activity obtained using the
Gal4 DBD alone. This level of stimulation is similar to that observed for hi 4-3-3 r| on
glucocorticoid receptor-dependent transcription (Wakui et al., 1997). This finding
suggests that 14-3-3 proteins have intrinsic potential to activate transcription when


67
were further increased (compare columns 6, 7, and 8 vs. 5, Fig. 2-12), implying that ftzQ
activity was squelched by high levels of TBP protein. However, this squelching effect
was not observed in transcription driven by the CaMV 35S promoter (compare columns
2, 3, and 4 vs. 1, Fig. 2-12), suggesting that optimal TBP concentrations vary between
different activators. This activator-dependent squelching by TBP is also observed in
other systems in vivo (Sadovsky et al., 1995). In general, the degree of squelching seen
in these experiments was not severe and did not alter conclusions regarding the
importance of TBP-TFIIB interaction in activated transcription.
The degree of transcriptional suppression by stirrup mutations of TBP varied
between the activation domains tested. For example, with the double mutation
E144R/E146R, transcription was reduced 16-fold for ftzQ (compare column 5 vs. 2, Fig.
2-7), 12-fold for VP 16 (compare column 5 vs. 2, Fig. 2-8), 11-fold for LpHSF8
(compare column 5 vs. 2, Fig. 2-10), 9-fold for VP1 (compare column 5 vs. 2, Fig. 2-
11), but only 4.5-fold for GAL4 (compare column 5 vs. 2, Fig. 2-9). A nearly 4-fold
difference in the degree of suppression was seen, with ftzQ being the most sensitive and
Gal4 AD the least affected.
Requirement for TBP-TFIIB Interaction Confirmed Using an Altered Specificity TBP
A TBP altered-specificity system was employed to further demonstrate the effect
of TBP stirrup mutations on activated transcription, free of possible interference by
endogenous TBP. The TATAA element of the GAL4/GUS reporter was mutated to
TGTAA so that activity would be dependent on expressed TBPm3, which is able to
recognize TGTAA in addition to TATAA (Strubin and Struhl, 1992). To demonstrate
the lack of recognition of the TGTAA motif by endogenous maize TBP, the empty


47
AtTFIIB recombinant proteins contained thirteen additional amino acid residues
(MASMTGGQQMGRS) at the N-terminal ends and two residues (El) at the C-terminal
ends followed by a stop codon. The first eleven residues are the T7-epitope
(MASMTGGQQMG).
Particle Bombardment in Maize Suspension Cells
The reporter and effector plasmid DNAs were precipitated onto gold particles by
the following procedure: 35 pi of DNA was mixed with 37 pi of gold (40mg/ml) in a 1.5
ml Eppendorf tube. 50 pi of 2.5 M CaCh and 20 pi of 100 mM spermidine were
pipetted onto the side wall of the tube separately, then immediately mixed with the gold
particles by vortexing for 20 sec. After spinning in a mini-centrifuge (5 sec), the
supernatant solution was pipetted out and discarded. The DNA-coated gold particles
were washed in 200 pi of 100% ethanol by sonication for 5 sec using a mini-sonicator
bath, then collected by centrifugation for 5 sec, and re-suspended in a final volume of 80
pi of 100% ethanol. For DNA-gold preparations, the amount of individual plasmid
DNAs remained constant between experiments as follows: 2.5 pg for Ubi/LUC as
internal control, 2.5 pg for the GUS reporter driven by the CaMV 35S minimal promoter
with or without upstream elements, 2.5 pg for Gal4 DBD fusion activators, and 10 pg
for T7, T7-TFIIB, T7-TBP (wt or mutants) effectors. The Ubi/LUC construct was driven
by a maize ubiquitin promoter previously described (Christensen and Quail, 1996). The
minimal promoter/GUS construct was under control of the CaMV 35S minimal
promoter (nt -46) (Odell et al., 1985), and used to measure the basal in vivo
transcription. The pBI221 vector was used to measure transcription activated by the wild
type CaMV 35S promoter in the 800 bp Hind III-BamH I fragment (Clontech). The


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TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS iii
LIST OF ABBREVIATIONS vii
ABSTRACT ix
CHAPTERS
1 LITERATURE REVIEW 1
The Pre-Initiation Complex 1
TFIID 5
The Holoenzyme 9
General Transcriptional Factor TBP 13
General Transcriptional Factor TFIIB 19
TBP-TFIIB Interaction 23
Interactions of TBP and TFIIB with Transcriptional Regulatory Proteins 26
Interactions with Activators 26
Interactions with repressors 31
Transacting Proteins 32
GAL4 32
VP16 34
Ftz 35
VP1 36
LpHSF8 37
The 14-3-3 Proteins 37
Rationale for the Present Study 38
2 CRITICAL ROLE OF THE TBP-TFIIB INTERACTION IN SUPPORTING
ACTIVATED TRANSCRIPTION IN PLANT CELLS 39
Introduction 39
Materials and Methods 46
Mutagenesis for AtTBP2 46
Particle Bombardment in Maize Suspension Cells 47
IV


36
contains the glutamine-rich activation domain ftzQ (aa 349-408), which also functions
as an activator when fused with the GAL4 DBD in yeast cells (Fitzpatrick and Ingles,
1989). In different protein-DNA complexes, ftz can either activate en (Fitzpatrick and
Ingles, 1989) or repress wg (Ingham et al., 1988) expression. The specific activity of ftz
can be regulated by its phosphorylation state (Dong et al., 1998; Krause and Gehring,
1989) and by its positive co-factor Ftz-Fl (Yu et al., 1997), which is a member of the
nuclear hormone-receptor superfamily. Transcriptional activation by ftzQ is believed to
be mediated by its interaction with TFIIB at the zinc-ribbon domain. The functionally
defective N-terminal region of TFIIB severely squelches the ftzQ activity (Colgn et al.,
1993), and the mutations, H17S and C33S, at the zinc-ribbon suppresses the squelching
and restores the ftzQ activity (Colgn et al., 1995).
VP1
VP1 is a maize transcriptional regulatory protein involved in seed maturation and
anthocyanin biosynthesis during seed development. It activates seed maturation-related
gene(s) expression such as Em, but represses seed germination-related genes such as a-
amylase genes (Hoecker et al., 1995). The protein contains a total of 691 residues with
the N-terminal 121 aa comprising the activation domain. This domain is rich in serine
and proline in addition to negatively charged amino acids and is predicted to form two
amphipathic a-helices similar to that of the VP 16 activation domain (McCarty et al.,
1991). This region can be deleted without affecting the repression function of VP1
protein (Hoecker et al., 1995). The B3 domain located at the C-terminal half of VP1 is
the DNA binding domain for the Sph element in the Cl promoter (Suzuki et al., 1997).
VP1 also activates transcription by the G-box element, presumably through protein-


98
In parallel experiments, interactions between hTFIIB and five additional
arabidopsis 14-3-3 iso forms were also observed (Fig. 3-IB). As with maize 14-3-3, the
arabidopsis iso forms specifically bound to the GST-hTFIIB beads, but not to the GST
beads (Fig. 3-IB). However, the binding affinities for hTFIIB appeared to vary between
isoforms as indicated by their differential retention on the GST-hTFIIB beads (compare
bound lanes vs. inputs lanes, Fig. 3-IB), with the highest binding efficiency seen for
isoform and the lowest for isoform oo. Taken together, these results suggest that the
14-3-3/hTFIIB interaction is a conserved phenomenon.
Conserved C-terminal Core of hTFIIB Binds Arabidopsis 14-3-3 Protein
Human TFIIB is known to interact with activator proteins in vitro and the two
repeat motifs located in the conserved C-terminus are important for binding to VP 16
(Roberts et al., 1993). To map the 14-3-3 binding domain(s) within TFIIB, a series of C-
or N-terminal deletion mutants were generated for hTFIIB and their potentials for
interactions with At 14-3-3 expressed in E. coli as GST fusions and immobilized on the beads. The free 14-3-3
protein was then incubated with the bead-immobilized hTFIIB constructs containing
approximately equal amounts of proteins. For the C-terminal deletion series of hTFIIB,
14-3-3 protein was retained by the construct containing aa 1-202 (lane 2, Fig. 3-2A), but
not to those containing aa 1 to 123 (lane 3, Fig. 3-2A) or aa 1 to 65 (lane 4, Fig. 3-2A).
Therefore, the hTFIIB region between aa 124 and 202 was required for 14-3-3 binding
(compare lane 3 vs. 2, Fig. 3-2A). For the N-terminal deletion series of hTFIIB, 14-3-3
protein was retained by immobilized GST-hTFIIB constructs containing aa 65 to 316
(lane 7, Fig. 3-2A), or aa 124 to 316 (lane 8, Fig. 3-2A), or aa 262 to 316 (lane 9, Fig. 3-


140
Vashee. S., and Kodadek. T. (1995). The activation domain of GAL4 protein mediates
cooperative promoter binding with general transcription factors in vivo. Proc.
Natl. Acad. Sci. USA 92, 10683-10687.
Verrijzer, C. P., Chen, J. L Yokomori, K., and Tjian, R. (1995). Binding of TAFs to
core elements directs promoter selectivity by RNA polymerase II. Cell 81, 1115-
1125.
Verrijzer, C. P., Yokomori, K., Chen, J.-L., and Tjian, R. (1994). Drosophila TAFII-
150: similarity to gene TSM-1 and specific binding to core promoter. Science
264, 933-941.
de Vetten. N. C., and Ferl, R. J. (1994). Two genes encoding GF14 (14-3-3) proteins in
Zea mays. Structure, expression, and potential regulation by the G-box binding
complex. Plant Physiol. 106, 1593-1604.
de Vetten, N. C., Lu, G., and Ferl, R. J. (1992). A maize protein associated with the G-
box binding complex has homology to brain regulatory proteins. Plant Cell 4,
1295-1307.
Wakui, H., Wright, A. P., Gustafsson, J., and Zilliacus, J. (1997). Interaction of the
ligand-activated glucocorticoid receptor with the 14- 3-3 eta protein. J. Biol.
Chem. 272, 8153-8156.
Walker, S. S., Reese, J. C., Apone, L. M., and Green, M. R. (1996). Transcription
activation in cells lacking TAFIIS. Nature 383, 185-188.
Wampler, S. L., and Kadonaga, T. (1992). Functional analysis of Drosophila
transcription factor IIB. Genes Dev. 6, 1542-1552.
Wang, W., and Shakes, D. C. (1996). Molecular evolution of the 14-3-3 protein family.
J. Mol. Evol. 43, 384-398.
Wang, Y., and Stumph, W. E. (1995). RNA polymerase II/III transcription specificity
determined by TATA box orientation. Proc. Natl. Acad. Sci. USA 92, 8606-
8610.
Washburn, K. B., Davis, E. A., and Ackerman, S. (1997). Coactivators and TAFs of
transcription activation in wheat. Plant Mol. Biol. 35, 1037-1043.
Weil, P. A., Luse, D. S., Segall, J., and Roeder, R. G. (1979). Selective and accurate
initiation of transcription at the Ad2 major late promotor in a soluble system
dependent on purified RNA polymerase II and DNA. Cell 18, 469-484.
Weinmann, R. (1992). The basic RNA polymerase II transcriptional machinery. Gene
Expr. 2, 81-91.


3
et ai, 1989; Maldonado et ai, 1990). This complex now functions as a platform for
docking of the polymerase to the promoter. This process is accomplished by three
interactions: TBP with CTD of the largest subunit of polymerase (Usheva et al., 1992),
the bridge of the RAP30 subunit of TFIIF between polymerase and TFIIB involving its
zinc-finger domain (Ha et al., 1993) and a direct interaction of polymerase with the
second direct repeat of TFIIB C-terminal core (Ha et al., 1993). As the final steps in PIC
assembly, TFIIE and TFIIH subsequently enter the complex to result in formation of a
complete functional PIC (Flores et al., 1992). In the form of the PIC, RNA polymerase
II is able to initiate RNA synthesis from a specific starting site with the aid of the
helicase activity of TFIIH to melt the DNA template in the presence of ATP (Holstege et
al., 1996).
Recently, a new approach has been taken to directly probe the structure of the
pre-initiation complex in vivo. The immunoprecipitation of nuclear extracts using
antibodies against either a general transcription factor or a subunit of the polymerase II
identified a multi-protein complex, the RNA polymerase II holoenzyme (Kim et al.,
1994b; Ossipow et al., 1995). The holoenzyme complex contains RNA polymerase II,
most general transcription factors and mediator proteins like SRBs. However, TFIID is
not included in holoenzyme (Kim et al., 1994b; Ossipow et al., 1995). This finding
suggest that in contrast to in vitro stepwise assembly, in vivo most of the general
transcription factors are pre-associated with RNA polymerase before assembly on the
promoter. Therefore, in vivo the PIC assembly is likely much simpler than that in vitro.
The whole process probably involves only two steps directly: the recognition of the core


112
Onion epithelial cells
o
Z)
_j
CO
=>
(5
0)
>
4-
TO
OH
7
6
5
4
3
2
1
0
Gal4/14-3-3
2ug DNA 20ug DNA
Amount of effector DNA used in transformations
Figure 3-9. Evaluation of transcriptional activity of GAL4 DBD/Atl4-3-3 <|>
in onion epithelial cells. Reporter, effector and internal control plasmids
were delivered into onion epidermal cells by particle bombardment. GUS
and luciferase enzymatic activities were determined after a 40 hr incubation
of transformed tissue at 25C in the dark. GAL4-dependent transcription was
calculated as a GUS/luciferase ratio of activity (nM MU/cpm). Bars
represent an average of three trials with standard deviation indicated.
Compared to Gal4 DBD alone, Gal4/Atl4-3-3 stimulated GUS expression
5-fold when high amounts of effector DNAs (20 pg) were used, but no
stimulation was observed with low amounts of effector DNAs (2 pg).


99
A.
binding to GST-hTFIIB
1% input 1-316 1-202 1-123 1-65 GST
1 2 3 4 5
-Atl4-3-3 Io/o binding to GST-hTFIIB
input 1-316 65-316 124-316 262-316 293-316 GST
6 7 8 9 10 11
-Atl 4-3-3 4
B.
helices: A1 B1 Cl D1 El + + + A2 B2 C2 D2 E2
Zn conserved
repeat 1
repeat 2
l
124
200 218
294 316
316
124 EZ
202 262 CZZmZZZZZI 292
hTFlIB
14-3-3
binding
+
+ +
+ + +
+
+ +
14-3-3 binding
domains
VP16 binding
domains
Figure 3-2. Identification of 14-3-3 binding domains within hTFIIB by deletion
analysis. A series of GST fused C- or N-terminal deletion mutants of hTFIIB were
tested for their abilities to interact with Atl4-3-3 <|>. Free 14-3-3 protein was
incubated in binding reactions identical to those used in Fig. 1 with equal amounts of
immobilized GST-hTFIIB deletion mutants. (A) Western blot probed with anti-Atl4-
3-3 antibody showing two regions of hTFIIB capable of interacting with the 14-3-3
protein. One is located from aa 124 to 202, the other from aa 262 to 292. (B)
Summary diagram of the results in (A) showing locations of binding domains for 14-
3-3 and VP 16.


48
GAL4/GUS reporter was controlled by the synthetic GAL4 binding sites as described
(Vemer and Gurley, in preparation). The effector constructs of either Gal4 DBD fusion
or T7-tag fusion were derived from the pBI221 vector as described (Vemer and Gurley,
in preparation). Within each experiment, the total DNA was kept constant by including
an "empty" vector having only the T7-tag coding sequence driven by the CaMV 35S
promoter. Constructs for reporter and effector plasmids are illustrated in figures 2-2 and
2-3.
The DNA-gold particles were delivered into log-stage maize suspension cells
using a Bio-Rad particle bombardment apparatus with set at 1100 psi. For each
bombardment, 5 pi of the well-suspended DNA-gold particles was pipetted onto the
carrier disk and allowed to air-dry. Cell samples were prepared as follows: 50 ml of cell
culture was poured into a 50 ml sterile centrifuge tube and the cells allowed to settle by
gravity to a volume of 5 to 7 ml. The extra medium was discarded, resulting in a
celkmedium ratio of 1:1 (v/v). The cells were then well suspended, and 300 pi was
pipetted onto a 2.5 cm diameter circle Whatman filter paper previously placed on an MS
phytogel plate (Murashige and Skoog, 1962). After particle bombardment, the filter-
immobilized cells were allowed to recover for 20 to 22 hr in the dark at 26C. The cells
were harvested by grinding using a mortar and pestle for 1 min in 300 pi of extraction
buffer containing 200 mM Na2HP04/NaH2P04, pH 7.8, 4 mM disodium
ethylenediamine tetraacetate (EDTA), 2 mM 1,4-dithiothreitol (DTT), 5% glycerol and
1 mg/ml bovine serum albumin (BSA). The supernatants were collected by
centrifugation for 15 min at 12,000 rpm and aliquots assayed for GUS and luciferase
activities.


30
this category are best described as weak activators. They are believed to be able to
support the initiation, but not the elongation of transcription (Blau et al., 1996). The
basis for this differential effect on initiation versus elongation is not understood at
present; however, the ability for an activation domain to interact with and, therefore, to
recruit TFIIH seems to be important for its activity in supporting the elongation of
transcription (Blau et al., 1996; Kumar et al., 1998).
Transcriptional activation by a particular activator may be enhanced or
suppressed by elevated levels of TBP or TFIIB proteins. When TBP or TFIIB is limiting
in a transcription system, the over-expression of TBP or TFIIB will enhance its
interaction with the activator and stimulate transcription. In contrast to this positive
effect, TBP and TFIIB also display a negative effect resulting from the squelching of
productive interactions with the activator. In this case, excess amounts of TBP and/or
TFIIB are unable to incorporate into the PIC and form nonfunctional complexes with the
activation domain to inhibit transcription. However, these effects often appear to be
activator-dependent. In the same transcription system in HeLa cells with multiple assays
for different activation domains, over-expression of TBP stimulated transactivation by
VP16, Tat, or estrogen receptor (ER), but inhibited transcription activated by Spl or NF-
1 (Sadovsky et al., 1995). The underlying mechanisms for these different responses by
activators are still unknown, but could be due to their differences in TBP affinity.
Sometimes, these positive or negative effects can also be cell-type-dependent as shown
by the vitamin D receptor (VDR), with which over-expression of TFIIB stimulated
VDR-dependent transcription in P19 embryonal carcinoma cells, but suppressed VDR
activity in NIH 3T3 cells, both in a dose-dependent manner (Blanco et al., 1995). It is


CHAPTER 1
LITERATURE REVIEW
The Pre-Initiation Complex
The protein-coding genes in eukaryotic cells are transcribed by RNA polymerase
II, which is a multi-subunit enzyme consisting of at least ten subunits in yeast (Woychik
and Young, 1990). Within the total enzyme complex, only the largest subunit RPB1 has
the catalytic activity. The C-terminal domain (CTD) of RPB1 is the regulatory domain
of the enzyme. The CTD contains a peculiar sequence of as many as 26 YSPTSPS
heptapeptide repeats in yeast (Lee and Greenleaf, 1989), 40 repeats in arabidopsis
(Dietrich et al., 1990) and 52 repeats in mouse (Corden et al., 1985). These repeated
sequences could be hypo- or hyper-phosphorylated at their serine and threonine residues
(Cadena and Dahmus, 1987). The phosphorylation/dephosphorylation of the CTD can
regulate transcriptional activity of RNA polymerase II. The enzyme is in a
transcriptionally active state when it is phosphorylated (Cadena and Dahmus, 1987),
while the dephosphorylation can arrest transcription (Dubois et al., 1994). However,
despite the inherent transcriptional function, RNA polymerase II alone is not sufficient
for gene-specific transcription because its highly nonspecific affinity with DNA results
in random transcription (Weil et al., 1979). For the proper transcription of a given gene,
the enzyme requires a group of proteins, the general transcriptional factors, to form the
pre-initiation complex (PIC) to specifically recognize the core promoter of the gene and
correctly initiate transcription (Kollmar and Farnham, 1993). The assembly of the PIC
1


31
not known whether the difference seen between the two cell line is due to participation
of different co-factors in transcription as suggested (Blanco et al., 1995).
Interactions with Repressors
Compared to activator proteins, functional mechanisms for repressor proteins are
much less clear. In general, however, interactions with PIC component(s), like TBP
and/or TFIIB, may also be crucial to the mechanism of transcriptional repression. As
opposed to activators, repressors can prevent PIC assembly by interacting with TBP
and/or TFIIB. For instance, the El A repression domain has been shown to bind to TBP
and disrupt TBP-TATA interaction. This effect can be reversed by TFIIB in vitro (Song
et al., 1997). The Dorsal Switch Protein (DSP1) inhibits TFIIA-TBP complex formation
by interacting with TBP (Kirov et al., 1996). The C-terminal repression domain of Eve
protein simply inhibits TFIIDs ability to recognize the TATA element by binding to
TFIID (Austin and Biggin, 1995). The Negative co-factor 2 (NC2) prevents TFIIB from
joining with the TBP-promoter complex by interacting directly with the promoter DNA
(Goppelt et al., 1996). The interaction of NC2 to DNA probably blocks the DNA surface
for the recognition by TFIIB. It appears that different repressors are able to prevent PIC
formation at different stages of the assembly; some blocking further assembly of the PIC
by masking protein-protein interactions, and others by interfering with DNA-protein
interactions. In addition, like co-activators, co-repressors may be required for some
repressors to function (Mermelstein et al., 1996). Overall, the details of interaction
within the PIC that distinguish proteins that activate versus those that repress
transcription are still poorly understood.