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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Plant Molecular and Cellular Biology thesis, Ph.D   ( 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.




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