Citation
Characterization and identification of a novel guanine nucleotide binding site on the bovine mitochondrial ribosome

Material Information

Title:
Characterization and identification of a novel guanine nucleotide binding site on the bovine mitochondrial ribosome
Creator:
Anders, John Claude, 1951-
Publication Date:
Language:
English
Physical Description:
xvi, 179 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Binding sites ( jstor )
Cattle ( jstor )
Gels ( jstor )
Irradiation ( jstor )
Ligands ( jstor )
Nucleotides ( jstor )
pH ( jstor )
Ribosomal proteins ( jstor )
Ribosomes ( jstor )
Binding Sites ( mesh )
Cattle ( mesh )
Department of Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Biochemistry and Molecular Biology -- UF ( mesh )
GTP-Binding Proteins ( mesh )
Guanine Nucleotides ( mesh )
Guanosine Triphosphate -- analogs & derivatives ( mesh )
Guanosine Triphosphate -- chemistry ( mesh )
Mitochondria ( mesh )
Research ( mesh )
Ribosomes ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 167-178.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by John Claude Anders.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
001581949 ( ALEPH )
24594102 ( OCLC )
AHK5867 ( NOTIS )
AA00006098_00001 ( sobekcm )

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Full Text









CHARACTERIZATION AND IDENTIFICATION OF A NOVEL GUANINE
NUCLEOTIDE BINDING SITE ON THE BOVINE MITOCHONDRIAL RIBOSOME










by
JOHN CLAUDE ANDERS


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


1990




CHARACTERIZATION AND IDENTIFICATION OF A NOVEL GUANINE
NUCLEOTIDE BINDING SITE ON THE BOVINE MITOCHONDRIAL RIBOSOME
by
JOHN CLAUDE ANDERS
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
1990


ACKNOWLEDGEMENTS
This work was made possible first by the love and support
of my wife, Valerie, and children, Jane, Benjamin, and
Katherine. Secondly, thanks are given to the U.S. Army which
provided the financial support for my graduate training. I
must thank Dr. Thomas W. O'Brien for his mentorship as my
major professor and the helpful guidance of the members of my
graduate studies committee. A special relationship often
develops between a student and their major professor and this
has been the case for me. Special thanks go to Dr. Nancy D.
Denslow who was instrumental in the initial discovery of GTP
binding to mitochondrial 28S ribosomes and also provided free
access to her work pertinent to this study and very helpful
insight and guidance. I must thank all the members of the
O'Brien laboratory for their patience in answering my
innumerable questions and especially to Mr. Michael Bryant who
often lead me through new techniques. Thanks go to fellow
graduate student Scott E. Fiesler, who collaborated with me in
the study of [a32P]ATP binding to mitochondrial ribosomes
presented in this study. Finally, thanks go to Michael Bryant
again, and to Mr. Bennie Parten of the protein core facility
in assistance with the chemical sequencing of mitochondrial
ribosomal proteins and peptides.


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS ix
ABSTRACT xv
CHAPTERS
1 INTRODUCTION 1
An Overview of GTP-Binding Proteins 1
Protein Synthesis 3
Conserved Amino Acid Sequences Among GTP-Binding
Proteins 6
Proteins Synthesis in Mitochondria 12
Proposal 16
2 NONCOVALENT BINDING OF MITOCHONDRIAL RIBOSOMES BY
NTPs 17
Introduction 17
Materials and Methods 19
Results 25
Discussion 33
3 PHOTOAFFINITY LABELING OF 28S RIBOSOMES WITH
8-AZIDO GTP 37
Introduction 37
Materials and Methods 43
Results 49
Discussion 72
4 PHOTOAFFINITY LABELING OF 28S RIBOSOMES WITH
[a P] GTP 79
Introduction 79
Materials and Methods 80
Results 81
Discussion 107


5 ISOLATION, IDENTIFICATION, AND PARTIAL AMINO ACID
SEQUENCE OF S5 AND OTHER RIBOSOMAL PROTEINS 112
Introduction 112
Materials and Methods 115
Results 128
Discussion 159
6 CONCLUSIONS AND FUTURE DIRECTIONS 167
REFERENCES 167
BIOGRAPHICAL SKETCH 179


LIST OF TABLES
Table page
1-1 Protein and rRNA Content of Ribosomes 14
2-1 Noncovalent Binding of Various [a32P]
Nucleotides to 28S Ribosomes 27
2-2 Competition for [a32P]GTP Binding to 28S
Ribosomes by Various Nucleotides 29
2-3 Noncovalent Binding of GTP and 8-Azido GTP to
28S Ribosomes 33
3-1 The Effect of Substituting KCL for NH4C12 and
the Lowering of the MgCl2 Concentration in the
Reaction Buffer on the Noncovalent Binding of
[a P]GTP to 28S Ribosomes 56
5-1 Summary of Peak Identification for RP-HPLC of
28S Ribosomal Proteins 144
5-2 Summary of Ribosomal Proteins and Peptides
Submitted for Gas Phase Amino Acid Sequence
Analysis 155
5-3 Summary of Amino Acid Sequence for Several
Bovine Mitochondrial Ribosomal Proteins and
CNBR Peptides of S5 156
5-4 Comparison of Amino Acid Sequence Found in S5
Corresponding to the E Site Found in Other GTP-
Binding Proteins Involved in Protein Synthesis. 162
v


LIST OF FIGURES
Figure page
1-1 Comparison of amino acid sequences from con
served binding regions, A,C,E, and G in GTP-
binding proteins 8
2-1 Noncovalent binding of [a32P]GTP, GMP, ATP,
UTP, and CTP to 28s bovine mitochondrial
ribosomes 26
2-2 Binding inhibition of [ mitochondrial ribosomes by various
nucleotides 28
2-3 Noncovalent binding of [a32P]8-Azido GTP and
[8- H]GTP to 28S bovine mitochondrial ribosomes 31
2-4 Binding inhibition of [a32P] 8-Azi do GTP to
bovine mitochondrial ribosomes by GTP 32
3-1 Generation of an aryl nitrene following uv
irradiation of 8-Azido GTP 40
3-2 Photoaffinity labeling of 28S ribosomes with
[a P] 8-Azi do GTP 51
3-3 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes witn [a P]8-Azido GTP.... 53
3-4 Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S
ribosomes with [a P] 8-Azi do GTP 58
3-5 Continued optimizatign gf the intensity and
duration of uv irradiation for photoaffinity
labeling of 28S ribosomes with [yP]8-Azido GTP 60
3-6 The effect of decreasing concentrations of MgCl2
on ohotoaffinity labeling of 28S ribosomes with
[y32P] 8-Azi do GTP 63
3-7 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with increasing
concentrations of [y P]8-Azido GTP 66
4-1 Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S
ribosomes with [a P]GTP 82
vi


4-2
4-3
4-4
4-5
4-6
5-1
5-2
5-3
5-4
5-5
5-6
5-7
5-8
Specificity of the photoinduced radiolabeling of
28S ribosomal proteins by [a P]GTP in the
presence of other nucleotides 85
Determination of the binding affinity of [aP]
GTP for the specifically labeled 38 xDa ribosomal
protein by photoaffinity labeling of 28S
ribosomes 90
Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with [a32P]GTP 94
Determination of the requirement for uv
irradiation to radiolabel 28S ribosomes with
[a P] ATP and [aSrP]GTP 100
Time course far the uv independent radiolabeling
of S4 with [a P] and [Y32P]ATP 104
Reverse phase high pressure liquid chromatogram
of 1.6 nmol of acetic acid extracted 28S
ribosomes resolved on a wide pore (300 A) silica
bonded butyl column 130
Two-dimensional PAGE analysis of 28S ribosomal
proteins eluted from a RP-HPLC butyl column
coelectrophoresed with a minimal amount of acetic
acid extracted 28S ribosomes 134
Immunoblot of 28S bovine ribosomal proteins with
an anti S5 monospecific rabbit sera 146
Immunoblot of 28S bovine ribosomal proteins with
an anti S5 monospecific rabbit sera 147
Immunoblot analysis of 28S bovine ribosomal
proteins separated by RP-HPLC with an anti S5
monospecific rabbit sera 150
Immunoblot analysis of S5 protein isolated by
RP-HPLC 152
Electrophoretic separation of cyanogen bromide
peptides of S5 154
Elucidation of a putative oxidized tryptophan
residue found in the 5 PTH amino acid residue
of the 13 kDa CNBR peptide of S5 158
vi i


5-9 Comparison of putative conserved regions found in
the amino acid sequence of CNBR peptides of S5 to
homologous regions found in other GTP-binding
proteins 161
Vi i i


ABBREVIATIONS

.... Angstrom, meter x 10'10
AA
.... amino acid
Ac
.... acetate
ACS
.... American Chemical Society
ADP
.... adenosine 5'-diphosphate
AR
.... autoradiogram
ATP
.... adenosine 5-triphosphate
ATZ
.... ani1inothiazolinone
AU
.... absorbance unit
8N3GTP
.... 8-Azido guanosine 5-triphosphate
BCIP
.... 5-bromo-4-chloro-3-indolyl phosphate
bp
.... base pairs
BME
.... 6-mercaptoethanol
C
.... degrees centigrade
%C
.... percent crosslinker (bisacrylamide)
C4
.... butyl
Ci
.... curie, 2.2 x 1012 dpm
cm
.... centimeter
cm2
.... centimeter squared
CNBR
.... cyanogen bromide
cpm
.... counts per minute
CTP
.... cytosine 5-triphosphate
cGMP
.... 3'-5'-cyclic guanosine monophosphate
cytoribosome.. cytoplasmic ribosome


d day
Da daltons
DEAE diethyl ami noethyl
DEPC diethylpyrocarbonate
dGTP 2'-deoxyguanosine triphosphate
DPTU N,N'-diphenylthiourea
DTT dithiothreitol
E. col i Escheri chi a col i
EDTA ethylenediaminetetraacetic acid
elF eucaryotic initiation factor
EF-Tu bacterial elongation factor-Tu
EF-Ts bacterial elongation factor-Ts
EF-G bacterial elongation factor-G
EF-1 eucaryotic elongation factor-1
f fraction
Fc constant fragment of IgG molecule
g force of gravity
GDP guanosine 5'-diphosphate
gm grams
GMP guanosine 5'-monophosphate
GMPPCP guanosine 5'-[B,Y~methylene]triphosphate
GMPPNP guanosine 5'-[6,Y_imido]triphosphate
GTP guanosine 5'-triphosphate
h hour
3H tritium
HPLC high pressure liquid chromatography
x


IF-1 bacterial initiation factor-1
IF-2 bacterial initiation factor-2
IF-3 bacterial initiation factor-3
IgG immunoglobulin G
Imm immune
ITP inosine 5'-triphosphate
Kd binding dissociation constant
kDa kilodaltons
Ki binding inhibition constant
m mi 11 i
M molar, moles per liter
mA milliamphere
Mr molecular mass
\i micro
ng microgram
nl microliter
nM micromolar
nW microwatt
mg milligram
min minute
mi toribosome.. mitochondrial ribosome
ml mi 11 i 1iter
mM millimolar
mmol mil limle
mRNA messenger ribonucleic acid
mt mitochondria
xi


MWM molecular weight markers
n number
NADP nicotinamide adenine dinucleotide phosphate
ng nanogram, grams x 10'9
nm nanometer, meter x 109
nmol nanomole, moles x 109
ID one-dimensional
NBT nitro blue tetrazolium
NC no competition
NEM n-ethylmaleimide
ND not detected
NTP nucleotide triphosphate
Org organic
P peak
32P 32phosphate
PAGE polyacrylamide gel electrophoresis
pg pi cograms, grams x 10'12
Pi phosphate
PI preimmune
PITC phenyl isothiocyanate
pmol picomole, moles x 10'12
POPOP l,4-Bis(5-phenyloxazol-2-yl)benzene
PPO 2,5-diphenyloxazole
Prep preparation
PTH phenylthiohydantoin
PVDF poly(vinylidene difluoride) membrane
XI 1


Rib ribosome
Res residue
RF release factor
RP-HPLC reverse phase-HPLC
RNA ribonucleic acid
rRNA ribosomal ribonucleic acid
S stained gels (used in figures)
S Svedberg
S5 small subunit ribosomal protein #5 (or 1 to 33)
SD standard deviation
sec seconds
SE standard error
SDS sodium dodecyl sulfate
SW sperm whale
%T percent total monomer
TBS tris buffered saline
TBSA tris buffered saline with sodium azide
TEA triethanolamine
TEMED N,N,N',N'-Tetramethylethylenediamine
TFA trifluoroacetic acid
TMA trimethyl amine
TP total protein
tRNA transfer ribonucleic acid
Tris-HCL tris-hydroxymethylaminomethane-hydrochloride
Triton X-100.. Octylphenoxypolyethoxyethanol
2D two-dimensional
xi i i


UTP uridine 5'-triphosphate
uv ultraviolet
V volt
X any amino acid
xi v


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
CHARACTERIZATION AND IDENTIFICATION OF A NOVEL GUANINE
NUCLEOTIDE BINDING SITE ON THE BOVINE MITOCHONDRIAL RIBOSOME
by
John Claude Anders
December, 1990
Chairman: Dr. T. W. O'Brien
Major Department: Biochemistry and Molecular Biology
Bovine mitochondrial ribosomes possess a single, high
affinity binding site for GTP and GDP on the small subunit.
This is in contrast to procaryotic and eucaryotic cytoplasmic
ribosomes which do not bind GTP directly but do so to soluble
protein factors which cycle on and off the ribosome. We used
a photoaffinity analog of GTP, 8-Azido GTP and well as GTP to
identify ribosome components associated with the GTP binding
site. 8-Azido GTP binding to the small subunit was saturable,
with an apparent dissociation constant (Kd) of 1.92 jiM,
approximately 126 times higher than that for GTP. Photolysis
of both the photoreactive analog and GTP resulted in the
specific labeling of a single ribosomal protein, S5.
Photolabeling of S5 by both [ was blocked competitively by the addition of excess GTP,
xv


demonstrating the labeling was site specific. Partial amino
acid sequence and analysis of this protein revealed two areas
of amino acid sequence having significant homology to
conserved sequences involved in GTP binding in other GTP-
binding proteins. This protein with a guanine nucleotide
binding site may participate in initiation complex formation
on mammalian mitochondrial ribosomes. These mitochondrial
ribosomes apparently employ a different mechanism for
initiation of protein synthesis than bacterial or eucaryotic
cytoplasmic ribosomes which are unable to interact directly
with GTP.


CHAPTER 1
INTRODUCTION
An Overview of GTP-Bindina Proteins
G-proteins are a central component of the cellular
machinery driving olfaction, vision, control of cell prolif
eration, cellular regulation, and protein synthesis (for
reviews see Stryer, 1986; Gilman, 1987; and Allende, 1988).
Their basic function in the first four instances is to mediate
signal transduction between the cell surface and the cyto
plasm. This process begins when a ligand binds to the extra
cellular portion of a receptor protein. Upon ligand binding,
a conformational change in the receptor allows binding of a
GDP binding protein to the cytoplasmic portion of the same
receptor. This results in the exchange of GDP for GTP on the
membrane bound G-protein. This exchange of guanine nucleo
tides is often mediated by protein exchange factors which are
an important site for regulation of these processes. This
exchange results in conformational changes in the G-protein
which results in dissociation of two subunits of the G-protein
heterodimer, commonly called GY, leaving an activated Ga-GTP
protein. Activated Ga-GTP now binds another membrane bound
acceptor protein such as cGMP phosphodiesterase, adenylate
1


2
cyclase, or phospholipase C, which results in catalytic
production of a second messenger molecule cGMP, cAMP, or
inositol triphosphate and diacyl glycerol f respectively. These
soluble, second messenger molecules complete the signalling
event by binding to tissue specific intracellular receptors.
In the case of second messengers cGMP and cAMP, they modulate
conductance of various ions through the plasma membrane.
Inositol triphosphate and diacyl glycerol bind to the
endoplasmic reticulum, resulting in release of intracellular
calcium, and to protein kinase C (PKC), forming an active PKC,
respectively.
Another feature of GTP-binding proteins is an intrinsic
GTPase activity which results in hydrolysis of GTP to GDP.
Following activation of the acceptor molecule, the GTP-binding
protein turns itself off by hydrolyzing the bound GTP to GDP
resulting in an altered conformation for the protein. This
results in a reduced affinity and dissociation from the
acceptor protein thereby inactivating the signaling event.
In protein synthesis, GTP-binding proteins function in
a different manner. There is no signaling across a cell
membrane upon ligand binding and no membrane bound G-protein
is involved, as described above. However, the G-proteins in
this system (initiation and elongation factors) do bind GTP.
Binding of GTP results in a conformational change in the
factor that allows it to bind to the ribosome and produce a
competent initiation or elongation complex leading to protein


3
synthesis. Hydrolysis of GTP, bound to these factors,
provides the energy to drive both initiation and elongation in
protein synthesis.
Protein Synthesis
G-Proteins are a key component in protein synthesis in
both procaryotic and eucaryotic systems. The components of
protein synthesis are analogous in both systems, differing
primarily in the increased complexity and numbers of accessary
proteins in the eucaryotic system. These accessary proteins
are involved in regulation of translation in the eucaryotic
system (for review see Moldave, 1985). Many features of
procaryotic ribosomes resemble that of eucaryotic ribosomes
therefore description of protein synthesis, will be largely
based upon the accumulated knowledge of translation in
procaryotic systems.
Translation of information encoded in messenger RNA
(mRNA) begins with the formation of a competent ribosomal
initiation complex. In E. coli. a GTP-binding initiation
factor, IF-2, binds GTP and initiator fmet-tRNA on the small
subunit of the ribosome forming a GTP-ternary complex. The
order of binding is random and IF-2 does not necessarily bind
initiator methionyl t-RNA prior to binding the ribosome
(Gualaerzi, 1977, and 1986). In conjunction with two other
protein initiation factors, IF-1 and IF-3, mRNA binds to
complete the initiation complex. Upon formation of the
complete initiation complex on the small subunit, the


4
intrinsic GTPase activity of IF-2 is activated and bound GTP
is hydrolyzed resulting in the release of GDP-bound IF-2, IF-
1, and IF-3 from the small subunit. The large subunit now
binds to the small subunit with the initiator tRNA held in
proper orientation with respect to the initiator codon
(usually AUG) of the mRNA to form a monosome to start
polypeptide elongation. IF-1 and IF-3 (which are not GTP-
binding proteins) are known to increase the binding affinity
of the GTP-ternary complex (Wintermeyer and Gualerzi, 1983)
and IF-3 also inhibits formation of monosomes (Noll and Noll,
1972).
Eucaryotic initiation factor, eIF-2, functions in a
manner nearly identical to IF-2. The major functional
difference is how GDP on the inactive initiation factor
exchange for GTP. The exchange of GDP for GTP occurs
spontaneously in procaryotes due to relatively low binding
affinities for both nucleotides. In eucaryotes the binding
affinity for GDP is typically several fold greater than for
GTP. An example of this is seen in Xenopus 1 eavis oocytes
(Carvallo and Al 1 ende, 1987) where GDP binds 50 times better
than GTP (Kd= 70 and 3800 nM for GDP and GTP, respectively).
The exchange of tightly bound GDP for GTP, on EIF-2, requires
a guanine exchange factor (GEF, Panniers and Henshaw, 1983)
which regulates initiation of protein synthesis in eucaryotes
(Ochoa, 1983).


5
Three elongation factors are required for protein
elongation, EF-Tu/Ts, EF-G in procaryotes and EF-1/1B, EF-2 in
eucaryotes. Two of these are GTP-binding proteins, EF-Tu, and
EF-G (EF-1, and EF-2 in eucaryotes) while EF-Ts (EF-1B in
eucaryotes) is an exchange factor. Both procaryotic and
eucaryotic elongation factors function in the same manner.
When GTP is bound to EF-Tu (or EF-1) a conformational change
occurs allowing binding of ami noacyl-tRNA to the factor (Crane
and Miller, 1974). Upon binding to the monosome and
positioning of the ami noacyl-tRNA in the P-site, the intrinsic
GTPase activity induces hydrolysis of GTP, and the GDP-bound
EF-Tu leaves the ribosome. The exchange factor, EF-Ts,
catalyses the exchange of GDP for GTP to reactivate the EF-Tu.
Elongation factor EF-Tu, bound with GTP can now bind the
ribosome. This induces the hydrolysis of GTP which provides
the energy to transfer of the ami noacyl-tRNA to the A site.
Once in the A site and the codon-anticodon pairing is correct,
the peptide chain is transferred from the P site (peptidyl
transferase reaction) to the amino group of the newly arrived
amino-acyl-tRNA. This generates a peptidyl-tRNA. Another
GTP-binding protein, EF-G catalyzes the translocation of the
peptidyl-tRNA from A to the P-site. The energy to drive
translocation is provided by the hydrolysis of GTP bound to
EF-G.
Termination of protein synthesis in procaryotes is not as
well worked out as are the initiation or elongation phases.


6
There are three release factors, RF-1, RF-2, and RF-3. RF-1
and RF-2 bind termination codons, UAA or UAG, and UGA,
respectively in mRNA (Tate and Caskey, 1974). The binding of
RF-1 or RF-2 to the ribosome stimulates the binding of the
third release factor, RF-3. This factor binds GTP and it
promotes the dissociation of release factors from the
ribosome, completing the synthesis of the nascent polypeptide.
A eucaryotic RF has been described from reticulocyte
preparations (Konecki et al., 1977) which also binds GTP and
has a GTPase activity.
The common elements seen in all the GTP-binding
initiation, elongation, and termination factors are that they
incur conformational changes upon binding GTP, that allow them
to mediate the binding of ami noacylated tRNAs or other protein
factors to the ribosome. Once completed they hydrolyze GTP to
GDP to provide the energy to release the factor or drive
protein synthesis.
Conserved Amino Acid Sequences Amona GTP-Bindina Proteins
Proteins that bind GTP and GDP comprise a diverse group.
The group includes the polypeptide initiation and elongation
factors, tubulin, transducin, adenylate cyclase receptor
proteins, and a family of ras proteins. The ras genes encode
a family of closely related proteins of approximately 21 kDa
and were initially identified in human lung and bladder
sarcomas (Scolnic et al., 1979; Shih et al., 1980; Papageorge
et al., 1982).


7
Halliday (1984) described four regions of homology among
various G-proteins whose amino acid sequences were known at
the time. The proteins were bacterial elongation factors (EF-
G and EF-Tu of E. coli). human c-H-rasl gene product (c-H-
ras). and yeast c-rassc gene product (figure 1-1). Van Meurs
et al. (1987) expanded this comparison to include the a-
subunits of both Ta rod and Ta cone transducin and the a-
subunits of GTP-binding proteins that modulate adenylate
cyclase, Gi and Gs. All sequence information for these
proteins were obtained from bovine sources. Recently,
several other notable G-proteins have been sequenced, a
bacterial initiation factor (IF2) and even a yeast
mitochondrial elongation factor (EF-Tumt, see figure 1-1 for
references). The same four regions of conserved sequence were
found in all these GTP-binding proteins from bacterial, yeast,
and mammalian sources and are shown in figure 1-1.
Two additional conserved sites for the mono-ADP
ribosylation by bacterial toxins were found in the a-subunits
of transducin and GTP-binding proteins that modulate adenylate
cyclase (for review see Ueda and Hayaishi, 1985). ADP-
ribosylation is a post-translational modification that
utilizes NAD as the donor of the ADP-ribose group and leads to
a decreased rate of GTP hydrolysis. This impaired intrinsic
GTPase activity leaves the GTP-binding protein in a
permanently activated (GTP-bound) state (Cassel and Selinger,
1977).


A
C
E
G
Gi
36
Go
36
Gs
43
T. rod
32
Ta cone
36
c-H-ras
6
Ras^
13
EF-fu
15
EF-G
14
IF-2
394
EF-Tu
51
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LVWGAGGV|GKSAlfr
IVWGGG GV GKSAIT
vjGjlGSvcgGKTTI
IGIS
VT
AHIDAGKT
MGHVCH
GTIGHViaHGKTT
198
199
221
198
194
55
62
78
85
442
115
MFDVGGQPSE
LFDVGGQFjSE
MFDVGGQRE
MFDVGGQPSE
MjFpVGGQgSE
ildSag
ILDIAG
IVDCPGHApY
FlDIPGlHvbF
FlLDIP
HJVDtdP1
216
217
239
212
216
76
83
98
106
463
136
EGVTjAfirncJV
EDVTATIFCV
NDVTAIIFV|V
EGVT CUFIA
EGVgCIIFpA
EG^LOVpAIN
EGFIpWSVT
DGAlfrj
DGA
VjVLW AAD
dgaiilwIaIat
264
265
287
260
264
112
118
130
137
494
167
IILFLNKKD
IILFLNKKD
VILFLNK@D
IVLFLNKKD
IVLFLNKKD
vlvIg^kBd
VWG-NKLD
IIVFLNKCD
KUD
KID
iwfvnkMd
oo
Figure 1-1 Comparison of amino acid sequences from conserved
binding regions, A,C,E, and G (Hal 1 id ay r 1984, and Van Meurs
et al., 1987) for GTP-binding proteins that modulate adenylate
cyclase Gia (inhibitory, Nukada, et al., 1986), Goa (brain; Van
Meurs et al., 1987), Gsa (stimulatory, Robisnaw et al., 1986,
and Michel et al., 1§86), T rod (Tanabe et al.. 1985,
Medynski et al., 1985, and Yatsunami and Khorana. 1985), Ta
cone (Lochrie et al., 1985, and Lerea et al., 198b), c-H-ras
(Capon et al., 1983), ras,c (i. cerevisiae. DeFeo-Jones et aTT7
1983), EF-Tu (E. coli. Jones et al., 1980, and Laursen et al.,
1981), EF-G (E. col i, Ovchinnikov et al., 1982), IF-2 (E.
col i. Saucerdot et al., 1984), and EF-Tumt (i. cerevisiae.
Nagata, et al., 1983). Boxes surround four or more residues
that are identical or conserved. Residues were grouped
according to Dayhoff (1978) conservative categories, C;
S,T,P,A,G; N,D,E,Q; H,R,K; M,I,L,V; F,Y,W.


9
Cholera toxin is a ADP-ribosyltransferase that modifies
the a-subunits of Gs (Cassel and Pfeuffer, 1978) and
transducin (Abood et al., 1982). The ADP-ribosylation site is
arginine specific, located at Arg174 in o-Transducin within the
conserved peptide, Ser-Arg-Val-Lys.
The second enterotoxin, pertussis toxin, modifies the a-
subunits of Gi(Bokoch et al., 1983 and 1984), Go (Hurley et
al., 1984), and also transducin (West et al.,1985). This
second ADP-ribosylation site is located within three residues
of the COOH-terminus of a-transducin at Cys347 (S-glycoside,
Hurley et al., 1984). This site resides within a conserved
peptide, Asp-Cys-Gly-Leu.
Various means have been employed to identify the
nucleotide binding sites of most of these proteins. The first
to be well resolved was elongation factor Tu (EF-Tu) from
Escherichi a coli. by means of X-ray crystallographic studies
(la Cour et al., 1985; Jurnak, 1985). De Vos et al. (1988)
determined the crystal of cellular ras p21 to the same
resolution as EF-Tu, 2.7 A. Essential features of both
structures are similar, only the structure of EF-Tu will be
discussed below.
Three domains of E.col i EF-Tu were found and the best
resolved was the GTP binding domain located at the NH2
terminal portion of the protein, comprising the first 200
residues. The GTP/GDP ligand site was found to be located at
the C00H terminal end of a sheet of four parallel R strands.


10
The guanine nucleotide was in the anti-conformation in respect
to the sugar and found in an unusual location, at the outer
edge of the domain, rather than within a hydrophobic pocket.
The guanine ring is partially buried in a cavity and appears
to be fixed in position by the side chains of four key
residues, Asn135-Lys-Cys-Asp. These residues represent a
highly conserved consensus sequence, of Asn-Lys-X-Asp, found
in region G (figure 1-1) of all GTP-binding proteins. The
ribose was exposed to the solvent. Asn135 and Asp138 lie near
the guanine and can bind the keto and amino substituents of
the guanine ring, respectively. Residues in both the E and G
region reside in two B-strands that interact with one side of
the guanine base (Jurnak, 1985, and la Cour et al., 1985).
Mutagenesis of Lys136, in conserved region G of bacterial
EF-Tu to an Arg or a Glu reduces the ability of the protein to
bind guanine nucleotides by about 20 or 100 fold, respectively
(Hwang et al., 1989). In the ras p21 protein, mutagenesis of
the corresponding Asp116 to Lys or Tyr in this same conserved
region abolishes GTP binding activity in this protein (Shih et
al., 1988).
In regions A and C (figure 1-1) are residues that
interact with the pyrophosphate group. Crystallographic
analysis by Jurnak (1985) and la Cour (1985) revealed that Mg2+
is located close to the B-phosphate of GDP, forming a salt
bridge to Asp80 of EF-Tu. This residue was found in the
conserved peptide, Leu-Asp-Thr-Ala-Gly, in region C. In the A


11
region, the sequence Gly-X4-Gly-Lys-Ser/Thr is found in all
GTP-binding proteins examined so far. Lysine24, in bacterial
EF-Tu, is at the NH2-terminus of a -helix which has been
postulated to form a positive dipole that partially
neutralizes the negative charge of one the phosphates (Hoi et
al., 1978, Jurnak, 1985). Additionally, Ohmi et al. (1988)
affinity labeled the corresponding Lys16 in the ras oncogene
product p21 with guanosine diphospho- and triphospho-
pryridoxals. These observation suggest that this residue in
the conserved sequence is located in the guanine nucleotide
binding site, close to the 6- or y-phosphate group of the
nuceotide.
Site directed mutagenesis in a family of closely related
genes, ras, which encode a group of homologous 21 Kda
proteins, has yielded additional information about guanine
interactions with these GTP-binding proteins. In region A the
conserved region Gly10-X4-Gly-Lys-Ser/Thr in ras p21 forms a
loop that interacts with the pyrophosphate group as described
above. Shih et al. (1988) mutated individually the Gly
residues at codons 10 and 15, to Val in ras p21 and found the
GTP binding was reduced by 33 and 1000 fold, respectively.
In Harvey and Kirsten sarcoma viruses, oncogenic ras
genes are found that contain a point mutation in this same
conserved region, resulting in the replacement of Gly12 for Val
(Taparowsky et al., 1982, Tabin et al., 1982, and Reddy et
al., 1982). Other transforming point mutations have been


12
identified and are discussed fully in the review by Barbacid
(1987).
Site directed mutagenesis of Gly12 in the cellular ras
proto-oncogene resulting in the substitution of any other
amino acid except proline for this residue conferred
transforming properties on the ras p21 gene product. The
authors postulated that a-helical structure in this region was
required for transformation (Seeburg et al., 1984). Insight
into the molecular events disturbed by this point mutation was
brought forth by McGrath et al. (1984). They found that this
single point mutation, resulting in the substitution of Gly12
by a Val, not only reduced GTP binding activity but also
impaired the intrinsic GTPase activity of ras p21 protein by
at least 10-fold. The transformation of the protoncogene
product, ras p21, was attributed to this lowered rate of
GTPase activity. Kinetic analysis of the hydrolysis of GTP by
p21 ras revealed that the mutant (Asp12) protein had a lowered
rate of hydrolysis and GDP release, in comparison to that of
the proto-oncogene (Val12), consistent with the reduced GTPase
activity found in this mutant (Neal et al., 1988).
Protein Synthesis in Mitochondria
The mechanism of protein synthesis in mammalian
mitochondria is poorly understood. Mammalian mitochondrial
ribosomes have been generally characterized as procaryotic in
nature due to some shared antibiotic sensitivities (Denslow
and O'Brien, 1974) and that both utilize f-Met-TRNA for


13
polypeptide initiation. However, a number of differences
exist. Susceptibility to lincosamies and macrolides is much
lower than in procaryotes suggesting some components of the
peptidyl transferase center are altered (Denslow and O'Brien,
1978). Additionally, mammalian mitochondrial ribosomes are
not sensitive to the antibiotic kirromycin, which stabilizes
the binding of the EF-Tu-GTP-aminoacyl-tRNA complex to the
bacterial ribosome, thereby inhibiting polypeptide elongation
(Schwartzbach and Spremulli, 1989).
In regard to their fine structure and physicochemical
properties, mitochondrial ribosomes differ considerably from
other kinds of ribosomes. The mass of the mitoribosome is
approximately the same as the procaryotic ribosome, but the
buoyant density is much less. This is due to the fact that
mitoribosomes contain only half the amount of rRNA and twice
the protein as bacterial ribosomes (Matthews, et al., 1982).
The number of ribosomal proteins is also much greater in the
mitoribosome (table 1-1). In addition, mi toribosomal proteins
do not appear to have any obvious homologue in the bacterial
or cytoribosome as identified by electrophoretic or immuno
logic cross-reactivity (Pietromonaco et al., 1984 and Matthews
et al., 1982). Based upon this evidence and functional
studies described below, O'Brien and Matthews (1976) have
assigned mitochondrial translation systems to four separate
classes (Protista, fungi, plants, and animals).


14
Table 1-1
Protein and rRNA Content of Ribosomes
Source
Ribosome
(S units)
Number of
Proteins
rRNA
(S units)
Bovine
28S"
33
12S
Liver
39Sb
52
16S
Mitochondria
55SC
85
Procaryotic
30S?
21
16S
E. coli
50Sb
32
23S, 5S
70SC
53
Mammalian
40S*
33
18S
Eucaryotic
60Sb
50
28S, 5.8S,5S
Cytoplasmic
80SC
83
¡Ribosome small subunit
Ribosome large subunit
cMonosome
The mechanism for binding mRNA in mi toribosomes appears
to differ from that of procaryotic and eucaryotic cytoplasmic
ribosomes. In procaryotes a leader sequence 5' to the
initiation codon (AUG) binds to a complementary sequence on
16S rRNA, called the Shine-Dalgarno (1974) sequence (Steitz
and Jakes, 1975). This mRNA binding site is absent in
mitochondrial mRNAs which often begin with the initiation
codon or have very short leader sequences which have no
complement on 12S rRNA (Clayton, 1984). Eucaryotic mRNAs also
lack any complementary binding to rRNA. However, they do have
a modified 5'-terminal modification, the m7G(5)ppp cap which
functions as a recognition signal (Shatkin, 1976) for a cap


15
binding protein. Once the cap binding protein binds the
modified mRNA it scans the 5'-untranslated mRNA leader for the
proper initiation codon. Though the mechanism for initiation
codon recognition in the mitoribosome is unknown, Denslow et
al. (1989) did discover that the major interaction of the mRNA
on the mammalian mitoribosome, covered a 30-nucleotide
segment. Also, the binding of authentic mitochondrial mRNAs
required homologous initiation factors.
Heterologous translocases and initiation factors from
several sources were tested to determine if they would
function with mammalian mitoribosomes. With few exceptions,
homologous factors were required to support i_n vi tro poly
(U)-dependent phenylalanine polymerization in mammalian
mitoribosomes. The only heterologous factor that has been
shown to support poly (U)-directed systems in animal
mitoribosomes was EF-Tu from a variety of sources (E. coll, B.
subtil is. and a protist Eualena gracilis. Eberly and
Spremulli, 1985). Denslow et al. (1988) also showed that
bacterial IF-3 could bind to small subunits of mitoribosomes
and inhibit formation of the monosome as it does in E. coli .
Bacterial EF-G (Denslow and O'Brien, 1979) and other trans
locases from gram-positive and gram-negative procaryotes,
protist, plants (chloroplast) and eucaryotic cytoplasmic
origins did not support i_n vi tro phenylalanine polymerization
(Eberly et al., 1985). This was surprising in view of the
fact that a postribosomal supernatant from E. col i would


16
function in a poly (U)-directed system in Neurospora crassa
mitochondrial ribosomes (Grandi etal., 1971). This suggested
that mammalian mitochondrial ribosomes may differ signifi
cantly from other types of mitochondrial ribosomes in their
interactions with soluble initiation and elongation factors.
The first reported isolation of any elongation factors
from mammalian mitochondria was EF-Tu/Ts complex from bovine
liver by Schwartzbach and Spremulli (1989).
Proposal
GTP binding proteins are an essential component in pro
tein synthetic machinery in procaryotic and eucaryotic cyto
plasmic systems. Though much is known about protein synthesis
in procaryotes and much more is being discovered about the
eucaryotic cytoplasmic system, little is known about protein
synthesis in mitochondrial systems. In a search for a puta
tive mitochondrial elongation factor, O'Brien et al. (1990)
discovered a unique GTP binding activity on the small subunit
of the mammalian mitochondrial ribosome. This GTP-binding did
not occur on soluble initiation or elongation factors, as is
the case for other ribosomal systems, but on the surface of
the mitochondrial ribosome, suggesting a novel mechanism for
initiation of protein synthesis. The objective of this
research was to identify the site of GTP binding on the small
subunit and further characterize this binding activity. Once
identified, the GTP-binding ribosomal protein was purified
and partial amino acid sequence was determined in an effort to
find any shared homologies to other GTP-binding proteins.


CHAPTER 2
NONCOVALENT BINDING OF MITOCHONDRIAL RIBOSOMES BY NTPs
Introduction
Guanosine-5'-triphosphate plays a key role in protein
synthesis in both procaryotic and eucaryotic systems. In both
systems GTP binds to soluble protein factors in a cyclic
manner to promote the initiation or elongation phases of
protein synthesis (Moldave, 1985, and Allende, 1988).
Elucidation of the translation process in mammalian
mitochondria has been difficult. The substitution of
heterologous translation factors from a variety of procaryotic
and eucaryotic sources generally fail to support poly (U)-
directed phenylalanine synthesis with mammal ian mitoribosomes.
Only EF-Tu from a variety of sources was capable of
functioning in mammalian mi toribosomal translation (Eberly et
al., 1985) as discussed above.
In an effort to isolate a putative mitochondrial EF-G
from bovine liver O'Brien et al. (1990) serendipitously found
that [8-3H] GTP binds to salt washed small subunits of
mammalian mitochondrial ribosomes. This is surprising due to
the fact that GTP-binding to activity in both procaryotic and
eucaryotic cytoplasmic translation systems occurs only on
17


18
soluble protein factors that cycle on and off the ribosome in
protein synthesis. This binding activity was resistant to
high salt washes (300 to 500 mM KCL) and independent of any
exogenous factors or fusidic which is known to stabilize EFG-
GTP complex on bacterial ribosomes. Factor independent
binding of GTP to procaryotic (E. col i 1 and eucaryotic
cytoplasmic (Bovine) ribosomes has never been reported and was
not observed when tested in this laboratory (Sonnega et al.
personal communication).
Although no function has as yet been associated with this
activity [8-3H]GTP was found to bind with high affinity (Kd=
15.3 nM) and in a stoichiometric manner to salt washed 28S
ribosomes (O'Brien et al., 1990). The large subunit (39S) and
intact monosomes (55S) did not bind GTP. The binding site was
specific for GTP and GDP and no other nucleotide tested could
compete with [8-3H]GTP binding to the small subunit. Binding
affinity for the 28S ribosome was equally high for GDP (Kd=18
nM).
Nonhydrolyzable analogs of GTP, GMPPNP and GMPPCP, also
bound to the small subunit, but with reduced affinity,
suggesting that binding site is sensitive to alterations in
the structure of the triphosphate side chain. Additionally,
[y32P] GTP was not hydrolyzed following binding to small
subunits. These findings suggest that salt washed ribosomes
do not posses an intrinsic GTP hydrolytic activity. This of


19
course did not rule out the possibility that GTPase activity
may require exogenous factors, as yet identified.
The following study will expand on the above information
in an effort to determine the specificity of this GTP-binding
activity. GTP-binding will be determined using standard
nitrocellulose filter binding assays and under various salt
conditions to determine the optimal ionic conditions for
binding.
Materials and Methods
Reagents
Guanosine[8-3H] 5'-Triphosphate was purchased as a
ammonium salt in a 50% ethanol solution at a specific activity
of 12.6 Ci/mmol and <99% purity (determined by manufacturer)
from Amersham Corporation (Arlington Heights, IL). Adenosine,
cytidine, guanosine, and uridine 5'[a32P] Triphosphate were
purchased as triethyl ammonium salts in aqueous solution at a
specific activity of 410 Ci/mmole from Amersham Corporation.
[a32P] Guanosine monophosphate was purchased also as a
tri ethylammonium salt from New England Nuclear (Boston MA) at
a specific activity of approximately 3000 Ci/mmol and of at
least 90% purity. 8-Azidoguanosine 5'[a32P] Triphosphate, was
purchased as a triethylammonium salt at a specific activity of
4.6 Ci/mmole from ICN Radiochemicals (Irvine, CA). All
unlabeled nucleotide-5'-triphosphates, guanosine di- and
monophosphates were Type I reagents, purchased as sodium salts
from Sigma Chemical Company. The NTPs were isolated from


20
Equine muscle free of vanadate ion and all other nucleotides
isolated from Yeast.
Mitochondrial Ribosome Preparation Solutions
Solution Formulation
A 0.34 M sucrose, 1 mM EDTA, 5 mM Tris-HCL, pH 7.5
B 0.26 M sucrose, 40 mM KC1 15 mM MgCl2, 0.8 mM
EDTA, 5 mM 2-mercaptoethanol (BME), 50 jiM
spermidine, 50 nM spermine, 14 mM Tris- HC1, pH 7.5
C 100 mM KC1, 20 mM MgCl2, 0.01% Triton X-100, 5 mM
BME, 20 mM triethanolamine (TEA), pH 7.5
D 40 mM KC1, 15 mM MgCl2, 0.05 mM EDTA 5mM BME 1.6%
Triton X-100, 0.005% diethylpyrocarbonate (DEPC),
10 mM Tris-HCl, pH 7.5
E 300 mM KC1, 40 mM MgCl2, 5 mM BME, 1.6% Triton X-
100, 0.005% DEPC, lOmM, Tris-HCl, pH 7.5
F 300 mM KC1 5mM MgCl2, 5 mM BME, 10 mM Tris-HCl, pH
7.5
G 0.34 M sucrose, 25 mM KC1, 5 mM MgCl2, 5 mM BME,
10 mM Tris-HCl, pH 7.5
Binding Assay Solutions
Reaction Buffer A: 100 mM KC1, 5 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer B: 10 mM NH4C1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer C: 100 mM KC1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME


21
fi-Scintillation Solution
Liquid Scintillation cocktail: 0.5% PPO and 0.05% (w/v) P0P0P
in toluene
Equipment
Beckman LS8000 6-liquid scintillation counter
Millipore 30 place vacuum manifold
Napco Vacuum oven, model# 5831
Preparation of Mitochondrial Ribosomes
Bovine mitochondrial ribosomes were prepared from bovine
liver as described previously (Matthews et al., 1982).
Mitochondria prepared from liver homogenates in buffer A were
treated with 100 |ag/ml digitonin to remove microsomal
membranes and then stored at -70C in buffer B until needed.
To isolate the ribosomes, thawed mitochondria were lysed in
1.6% Triton X-100 and clarified by centrifugation at 10,000
rpm for 45 min in a Beckman JA-10 rotor at 4C. Bovine
mitoribosomes were concentrated by absorption to DEAE
cellulose in buffer D and eluted with high salt (buffer E).
The crude mitoribosomes eluted from the DEAE cellulose were
layered on a 34% sucrose cushion (20ml) prepared in buffer C
and centrifuged at 35,000 rpm for 24 h in a Beckman type 35
rotor. The pellets were resuspended in buffer C, treated with
1 mM puromycin to remove nascent polypeptides, and centrifuged
in 10-30% sucrose density gradients prepared in buffer F to
yield 28S and 39S subribosomal particles. Dissociation of
subribosomal particles in high salt buffer (buffer F with 300


22
mM KC1) removed any soluble initiation or elongation protein
factors. Subribosomal particles were recovered from gradient
fractions by centrifugation at 100,000 x g in a Beckman Ti 60
rotor for 15 h, resuspended in buffer G and stored at -70C
until needed.
Binding of GTP to 28$ Ribosomes and Analysis bv Millipore
Filter Assay
Binding activity was determined by means of a Millipore
filter assay. In this assay, ribosomal subunits with bound
radiolabeled NTP were absorbed to nitrocellulose and washed to
remove unbound NTP. The reaction mixture consisted of a fixed
concentration of ribosomal protein (0.1 nM) with varying
concentrations ( 0.05 to 5 nM) of radiolabeled NTP, in
reaction buffer (A, B, or C). The specific activity of the
radiolabeled NTP (specific activity was typically 410 Ci/mmol)
was reduced by dilution with the appropriate unlabeled NTP
just prior to use. The final volume was adjusted to 50 jil
with reaction buffer in a 1.5 ml conical plastic eppendorf
tube and incubated for 5 min on ice. The reaction was stopped
by the addition of 0.5 ml of ice cold reaction buffer and
immediately transferred to a wetted nitrocellulose filter.
The filter was in a Millipore 30 place manifold under vacuum
to remove the aqueous buffer. Another 0.5 ml volume of
reaction buffer was used to rinse out the reaction tube and
also passed through the nitrocellulose filter. The filters
with bound protein were immediately washed with an additional


23
8 ml of ice cold reaction buffer also under vacuum. The total
time for the wash steps was approximately 15 seconds.
The nitrocellulose filters were placed in empty 20 ml
scintillation vials and dried under vacuum and at 70C for
approximately 1 h. The dried filters were solubilized in 5 ml
of scintillation cocktail solution (0.5% PPO, and 0.05% w/v
P0P0P in toluene). The filters were counted in a b-
scinti11ation spectrometer (Beckman, model #LS8000) at a
counting efficiency of 21% and 88% for 3H and 32P,
respectively. To determine the extent of nonspecific binding
of radiolabeled NTPs to nitrocellulose, control incubations
were done in an identical manner, but in the absence of
ribosomes. The amount of radiolabeled NTP that nonspecific-
ally bound to the filter was then subtracted from the corres
ponding treatment. This nonspecific binding was typically <
1% of the experimental treatments. Additionally, total
amounts of radiolabeled NTPs (the same concentration and
specific activity of radiolabeled NTP used in the experiment)
were determined for each experiment. This was done by pipet
ting an appropriate amount of radiolabeled NTP onto a wetted
nitrocellulose filter without washing, drying, then counting.
Binding data were plotted the aid of personal computer
using the Enzfitter Kinetics program by Leatherbarrow (1987).
Data was expressed as pmol of radiolabeled NTP bound per pmol
of ribosomes (R) versus the concentration of free NTP (C)


24
using an equation for ligand binding from Edsall and Wyman
(1958) as shown below:
R = n (C) / Kd + C
where: C= concentration of free ligand; Kd= dissociation
constant of ligand; and R= pmol ligand bound/ pmol ribosome
The binding data was linearly transformed by Scatchard
analysis (Scatchard, 1949) by the following equation:
R/C = -1/Kd (R) + n/Kd
where: R= pmol ligand bound / pmol ribosome; Kd= binding
dissociation constant* n= number of binding sites; C=
concentration of free ligand
Binding inhibition studies were done by incubating 0.1 jiM
28S ribosomes with increasing concentrations of (0.5 to 10 nM)
unlabeled NTP for 1 min in reaction buffer for 1 min on ice
under dim light. [a32P]GTP is then added at a saturating
concentration (0.1 *iM) for 5 min under the same conditions.
The samples are then washed and counted as described above for
the amount of [a32P]GTP that is bound to the ribosome in the
presence of increasing concentrations of NTP.
Binding inhibition data were plotted as percent of
control (Y) versus inhibitor concentration with the aid of
personal computer using the Enzfitter Kinetics program by
Leatherbarrow (1987). A modified equation for binding inhibi
tion (Cantor and Schimmel, 1980) was used and is shown below:
Y= l-[ l-r(I/Ki)/ 1+r[1+I/Ki] ]xl00
where: r= Kd/A=constant (Kd for GTP= 15.2 nM and A=
concentration of GTP); Y=l-vi/vo (vi/vo = percent inhibition).


25
Results
Binding of NTPs to 28S Ribosomes
O'Brien et al. (1990) found that [83H]GTP binds with high
affinity to 28S ribosomes and appears to be specific for GTP
and GDP only. To further investigate the nucleotide
specificity of binding for the small subunit, a number of NTPs
were tested for their ability to bind to ribosomes and also
their ability to compete for [a32P]GTP binding to the 28S
ribosome. Millipore filter binding assays (figure 2-1 and
table 2-1) confirmed that [a32P]GTP did bind to 28S ribosomes
with high affinity (Kd of 14.3 nM) and in nearly unit
stoichiometry which agrees with the result (Kd = 15.3 nM) of
O'Brien et al. (1990). The only other [a32P]NTP tested that
was found to bind to this ribosome in a stoichiometric manner
was GMP. GMP binds with approximately 800 times lower
affinity to the 28S ribosome than GTP. [a32P]ATP, UTP, and CTP
did not bind to the 28S ribosome to any appreciable degree.
Binding inhibition studies were done to determine which
nucleotides could displace [a32P]GTP from the 28S ribosome.
Denslow et al. (personal communication) found that of the NTPs
(GDP, ATP, CTP, and UTP) tested only GDP was able to compete
for [a32P] GTP binding. In this experiment additional
nucleotides were tested to determine the structural
requirements for this GTP binding activity. In agreement with
the results of O'Brien et al. (1990), GDP did compete strongly
for [a32p]GTP binding to the ribosome with a resulting Ki of


26
UJ
2
o
CO
o
99
cu
Q
Z
3
O
m
Figure 2-1 Noncovalent binding of (a P]GTP, GMP, ATP, UTP,
and CTP to 28S bovine mitochondrial ribosomes. Binding of the
radiolabeled nucleotides to the ribosomes was determined by
the Mi 11ipore filter binding assay. The same ribosomal
preparation was used for all nucleotides and the assay was
done in reaction buffer A (100 mM KCL, 5 mM MgCk, 10 mM Tris-
HC1, pH 7.4, and 1 mM BME) as described in Methods. The
initial specific activity of the [a P]NTPs was 410 Ci/mmol and
was diluted approximately 160 fold with unlabeled NTP to a
specific activity of 2.15, 1.67, 3.39,and 3.07 Ci/mmol for
GTP, ATP, CTP, and UTP, respectively just prior to use.
[a P]GMP was initially at a specific activity of 3000 Ci/mmol
and diluted with GMP to a specific activity of 10.2 Ci/mmol
just prior to use. The concentration of the ribosomes was 0.1
nM and the radiolabeled nucleotides varied from 0.05 to 10 uM.
Each data point represents the mean of duplicate samples
except for [aP]GTP where individual data points are shown
without averaging. Refer to table 2-1 for Scatchard analysis
of this data and to Methods for additional experimental
detai 1s.


27
9.2 nM (figure 2-2 and table 2-2) suggesting that GTP and GDP
bind to the same site on the 28S ribosome equivalently.
Additionally, ITP and GMP were able to compete to a lesser
extent, with binding inhibition constants (Ki) of 124 and 1140
nM, respectively.
Table 2-1
Noncovalent Binding of Various fa32Pl Nucleotides to 28$
Ribosomes
Nuceotide
(Kd SE)a
(nM)
Number Binding
(n SE)
GTP
14.1 2.3
0.80 0.02
GMP
11,500 1,300
1.00 0.06
ATP
77 44
0.09 0.01
CTP
ND
ND
UTP
1,200 990
0.07 0.02
aScatchard analysis of noncovalent binding of [a P]NTP and
[a P]GMP to 28S ribosomes shown in figure 2-1. Data
represents binding dissociation values (Kd) and standard error
(SE) from a single binding experiment. ND: Not detected
The ionic conditions used in which these binding assays
were modified from those used in earlier work by O'Brien et
al. (1990) in order to determine optimal stringencies for the
study of this GTP binding activity. The original work by
O'Brien et al. (1990) used reaction buffer B (10 mM NH4CL, 20
mM Mg2CL2, 10 mM Tris-HCl, pH 7.4, and 5 mM BME) which was


28
o
cu
t
z
o
o
J
o
ti:
L
CL
Figure 2-2 Binding inhibition of [a32P]GTP to bovine
mitochondrial ribosomes by various nucleotides. Displacement
of bound [a P]GTP (0.l|iM) from ribosomes (0.1 jiM) by
increasing concentrations (0.5 to 10 jiM) of competing
nucleotide was done in reaction buffer A as described in
Methods. The [a P]GTP was diluted with GTP to a specific
activity of 21. b and 14.8 Ci/mmol just prior to use for two
separate binding inhibition experiments (GDP, GMP,
dGTP,ATP,CTP, and GDP, UTP, ITP respectively). The same
preparation of ribosomes and chemical lot of [a PlGTP was
used in both experiments. The binding of [a P]GTP to
ribosomes in the presence of competing unlabeled nucleotide
was compared to binding in the absence of the competing
unlabeled nucleotide and represented as percent of control.
The data are single data points and each curve represents a
single experiment except for GDP where each data point is a
mean of two experiments conducted on consecutive days. Refer
to table 2-2 for analysis of binding inhibition for each
nucleotide described above.


29
Table 2-2
Binding Inhibition for fcx32Pl GTP Binding to 28S Ribosomes bv
Various Nucleotides
Nucleotide
Ki SEa
(nM)
GDP
ITP
GMP
dGTP
CTP
UTP
ATP
9.2 1.0
124 19
1,140 270
NC
NC
NC
NC
aBinding inhibition (Ki) of various nucleotides to compete for
aP]GTP binding to the 28S ribosome. Data represents values
Tom a single binding experiment and standard error, shown in
figure 2-2, for a single binding inhibition experiment.
NC: No binding inhibition
developed by Bodley (Bacca, et al., 1976) for the study of GTP
binding to bacterial EFG. Reaction buffer A (100 mM KC1, 5 mM
MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME) was developed
during the progress of the present study and was used in the
above binding and inhibition binding studies. The development
of these ionic conditions will be described later in this
paper. At this point it suffices to say that GTP binds
specifically to the 28S ribosome and quantitatively in the
same manner over a wide range of ionic conditions and
stringencies.


30
Both r
8-3HlGTP and ro32Pl 8-Azi do GTP Bind to the Same Site on
the 28
S Ribosome
In order to determine if the 8-Azido analog of GTP would
be a suitable photoaffinity probe for labeling the GTP binding
site on the 28S ribosome, filter binding and binding
inhibition studies were performed. In an experiment directly
comparing binding of [a32P]8-Azido GTP and [8-3H]GTP to 28S
ribosomes, the GTP analog did bind to the small subunit with
an apparent dissociation constant of 1.35 jiM (figure 2-3).
The dissociation constant for [8-3H]GTP was 18.1 nM which is
in close agreement with that found by O'Brien et al. (Kd=15.3
nM, 1990) and both bound in unit stoichiometry.
A mean dissociation factor for [a32P]8-Azido GTP and
[a32P]GTP have been determined (table 2-3). These values were
1.92 0.84 nM and 15.2 8.64 nM, respectively. Both
compounds bound to the ribosome in a stoichiometric manner
(1.2 0.4 and 0.84 0.17 sites occupied per ribosome,
respectively). These data suggest that the GTP analog binds
to the 28S ribosome with an affinity approximately 125 times
less than GTP. These mean Kd values for 8-Azido GTP and GTP
will be used throughout the remainder of this paper.
GTP was found to compete with [a32P] 8-Azi do GTP for
binding to the 28S ribosome with an apparent Ki of 106 nM
(figure 2-4). This suggests that GTP and its photoreactive
analog bind to the same binding site on the 28S ribosome.
These data imply that [a32P]8-Azido GTP could be used as a
photoaffinity probe for the identification of the GTP binding
site on the 28S ribosome.


31
0.0 0.2 0.4 0.6 0.8
FREE NTP (X 106M)
Figure 2-3 Noncovalent binding of [a 2P]8-Azido GTP and [8-
HJGTP to 28S bovine mitochondrial ribosomes. Binding of the
radiolabeled nucleotides to the ribosomes was determined by
the Mi 11ipore filter binding assay as described in Methods.
The same ribosomal preparation was used for both nucleotides
and the assay was done in reaction buffer B (10 mM NH,C1, 20
mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME) as described
in Methods. The specific activity of [8-H]GTP and [a P]8-
Azido GTP was 3.1 and 0.98 Ci/mmol, respectively. Each data
point represents the mean of duplicate samples. Scatchard
analysis for the binding of GTP and its 8-Azido analog to 28S
ribosomes revealed an apparent Kd of 18.1 and 1.35 jiM,
respectively.


32
o
o
X
o
Od
o
o
z
L
O
Od
J
Q_
INHIBITOR CONCENTRATION (X 106M)
Figure 2-4 Binding inhibition of [a32P]8-Azido GTP to bovine
mitochondrial ribosomes by GTP. Displacement of saturating
concentration (1.67 jiM) of [a P]8-Azido GTP from 0.083 jiM
ribosomes by increasing concentrations (0 to 16.7 ^M) of GTP
was done in reaction buffer B as described in Methods. The
specific activity of [a P]8-Azido GTP was 0.98 Ci/mmol The
same preparation of ribosomes and lot of [a P]8-Azido GTP was
used in this experiment and the one described in figure 2-3.
The binding of [a P]8-Azido GTP to ribosomes in the presence
of competing unlabeled nucleotide was compared to binding in
the absence of the competing unlabeled nucleotide and
represented as percent of control. The data are mean values
from duplicate samples and each curve represents a single
experiment. GTP was found to compete for [a P]8-Azido GTP
binding to 28S ribosomes with an apparent Ki of 106 nM.


33
Table 2-3
Noncovalent Binding of GTP and 8-Azido GTP to 28S Ribosomes
Nucleotide Reaction (Kd SD) Binding Sites
Bufferc (nM) (n SD)
[8-3H & a32PlGTPa A 15.2 8.64 0.84 0.17
[a32P]8-Azido GTPb B 1,920 840 1.20 0.40
aData for the binding of [8-3H] and [ are the mean and standard deviation of / experiments.
bData for the binding of [a32P] 8-Azi do GTP to 28S ribosomes are
the mean and standard deviation of 3 experiments.
formulations for reaction buffers A and B are listed in
Materials
Piscussion
There is one (stoichiometric) nucleotide binding site on
the 28S ribosome and it is specific for GTP and GDP.
Additionally, O'Brien et al. (1990) suggested that the binding
may occur on the interface of the ribosomes because [8-3H]GTP
did not bind to 55S monosomes. However it is not ruled out
that monosome formation induces a conformational change in the
GTP binding site, preventing binding of the nucleotide.
Only GTP and GDP bind the small subunit, and they do so
equally and with high affinity. Most GTP binding proteins
bind GDP more tightly than GTP, requiring an exchange factor
in order to regenerate the active GTP-bound protein. However


34
there are several instances where GTP-binding proteins do have
equal affinities for both GTP and GDP, such as the photo
receptor G-protein of frogs (Robinson et al., 1986), E. coli
EF-G (Kaziro et al., 1978), and p21 ras protein (Shih et al.,
1988).
The structural requirements for nucleotide binding in the
GTP/GDP binding site on the 28S ribosome were elucidated from
binding and binding inhibition assays as described above. No
difference were seen in binding affinities for GTP vs GDP
suggesting that the loss of the y-Phosphate does not perturb
binding. However the loss of the 6-phosphate as seen in GMP
does decrease binding affinity by at least 100-fold (table 2-
2). Additionally, O'Brien et al. (1990) reported that
nonhydrolyzable analogs of GTP, GMPPNP and GMPPCP, do bind the
28S ribosome, but with much reduced affinity. This was
attributed to alteration of the PB-X-P angles in these analogs
perturbing the binding interactions with the pyrophosphate
group (Yount, 1971).
Substitutions on the guanine ring also appear to be
poorly tolerated (see figure 3-1 for ring numbering system).
The substitution of the purine by a pyrimidine ring as seen
for CTP and UTP completely abolishes the ability of the
nucleotide to bind to or compete for [a32P]GTP binding to the
28S ribosome (table 2-1 and 2-2, respectively). This suggest
that functional groups on the base are misaligned in the
single ring base, preventing interactions with the binding


35
site. Inosine triphosphate did compete partially with
[a32P]GTP binding to the small subunit suggesting that the loss
of the amino group at C2 of GTP can be tolerated to some
extent. However the loss of the same amino group and the
substitution of an amino for the keto group at C6 as in ATPf
abolished all binding activity, suggesting that the keto group
was required for binding. Both the keto and amino group of
the guanine base have been shown in crystallographic studies
to bind to Asn and Asp residues, respectively in the highly
conserved G region (figure 1-1) in bacterial EF-Tu and ras p21
(Jurnak, 1985, la Cour et al., 1985, and De Vos, et al.,
1988).
Alterations of the ribose moiety are not tolerated
either. dGTP did not displace [a32P]GTP from the binding site,
indicating the importance for the hydroxyl group at C2, on the
ribose. O'Brien et al. (1990) also reported that periodate
cleaved GTP did not bind the small subunit suggesting that any
distortion in the conformation of the ribose affects its
binding. Additionally, these investigators found that n-
ethylmaleimide (NEM), a sulfhydryl modifier had no effect on
GTP binding activity, suggesting that a cysteine residue is
not located near critical GTP-binding residues.
The addition of the azi do (N3) moiety at C8 of the guanine
base reduces the affinity of this GTP analog for the small
subunit (Anders et al., 1989). This could be attributed to
stearic hindrance of the planar azido group and a change to


36
the syn conformation of the base preferred by 8-azido purines
(Colman, 1983). In crystallographic studies of EF-Tu and ras
p21 the GTP molecule resides in the GTP binding pocket in the
anti conformation (Jurnak, 1985, la Cour et al., 1985, and De
Vos et al., 1988) suggesting this conformational change could
contribute, at least in part, to the lowered affinity of this
GTP analog for the 28S ribosome.
This nucleotide binding activity on the ribosome, which
is highly specific for GTP and GDP, is consistent with other
GTP-binding proteins. GTP-binding proteins bind GTP in the
nM to low *iM concentration range and GDP commonly in the low
nM range (see reviews by Stryer, 1986; Gilman, 1987; and
Allende, 1988). They also have a single nucleotide binding
site that is specific for GTP and GDP. In a comparison of
nucleotide specificity, number of binding sites, and binding
affinity, 28S ribosomes bind GTP and GDP in a similar manner
as reported for other GTP-binding proteins. The only possible
difference is that an intrinsic GTPase activity has not yet
been observed on the salt washed 28S ribosomes. This does not
rule out the possibility that an exogenous factor(s), as yet
unidentified, may be required for GTP hydrolysis.


CHAPTER 3
PHOTOAFFINITY LABELING OF 28S RIBOSOMES WITH 8-AZIDO GTP
Introduction
Photoaffinitv Labeling of GTP-Bindina Proteins
Labeling receptor molecules with photoreactive analogs of
effector molecules has become a popular method in elucidating
the mechanisms by which their natural analogs regulate
biological phenomena. The chemical approach to receptor site
labeling is still used but suffers from the necessity to
contrive a molecule that can bind in sufficient concentration
to ensure preferential reaction with suitable residues that
can react with the modified ligand. Most of these reagents
are electrophiles which leads to selective incorporation that
may not reveal the residues in close proximity to the binding
site of the 1igand.
Photoreactive ligands can be constructed with addition of
one or just a few photosensitive constituents available today.
The photoreactive moieties commonly used are either diazo- or
azido-derivatives of their natural ligands (i.e.,ATP, cAMP,
GTP, cGMP etc.) and which should bind their receptor, assuming
sufficient latitude existed in the ligand-receptor recognition
37


38
process. They remain unreactive until irradiated with light
by the investigator. Irradiation of the diazo- or azi do-
purine analog provides the energy to form a reactive carbene
or nitrene radical, respectively, that may form a covalent
bond to residues at or near the binding site in a nonselective
manner. These reactive intermediates do display some
electrophilic character. However, -carbonyl carbenes and
aryl ni trenes, which have been used extensively to identify
nucleotide binding proteins, display wide chemical reactivity
by being able to insert into C-H bonds and other heteroatomic
bonds (Cooperman, 1980).
The ability to control the labeling event and provide
relatively nonselective incorporation of modified ligands has
made this a popular technique for the identification of purine
binding proteins. With the development and commercial
production of 8-Azido analogs of ATP, cAMP, GTP, and cGMP by
the methods of Haley, (1976), and Czarnecki et al. (1979), the
use of these reagents has increased. 8-Azido GTP was used in
this study. Therefore, the following discussion will be
limited to the nitrene, and not carbenes, even though the two
are of the same isoelectronic specie (six valence electrons)
and their chemistries are expected to be qualitatively
similar. Some examples are; azido analogs of GTP have been
used as photoaffinity probes to study GTP-subunit interaction
in adenylate cyclase (Pfeuffer, 1977), tubulin (Potter and
Haley, 1983), transducin (Kohnken and Me Connell, 1985), and


39
bacterial EF-G (Girshovich and Kurtskhalia, 1979). The number
of applications of these reagents is vast and the reader
should consult reviews by Bayley and Knowles, 1977; and
Chowdry and Westheimer, 1979).
The photoreactive azi do compounds remain unreactive in
the absence of light, thus allowing the investigator to bind
the ligand to the binding site before activating the
photoreactive reagent. Once bound, the sample is irradiated
with uv light and a molecule of N2 is photoeliminated and a
reactive nitrene forms (figure 3-1). This nitrene is highly
reactive (Turro, 1980; Bayley and Knowles, 1977; and Chowdhry
and Westheimer, 1979) and it can insert into heteroatomic
bonds of residues at or near the binding site, in a
nonselective manner (Richards and Konigsberg, 1977). The
type of bond formed is dependent upon the nature of the
nitrene. The nitrene comes in two forms; a very reactive
singlet nitrene that prefers to insert into 0-H and N-H bonds,
and the less reactive triplet nitrene that prefers C-H bonds
(Bayley and Knowles, 1977). The nitrene atom in both species
is linear in shape and the orbital occupancy is two
filled(sp2)4 orbitals for the singlet, and one filled (sp2)2
and two p orbitals (px)1 and (pz)1 for the triplet nitrene
(Turro, 1980). The singlet nitrene can covalently attach
itself in a single step reaction such as the addition to a C=C
bond, nucleophilic attack of N-H or 0-H bonds, or direct
insertion into C-H bonds in the backbone or si dechai ns of


40
AZIDE FORM
NITRENE FORM
Figure 3-1 Generation of an aryl nitrene following uv
irradiation of 8-Azido GTP. Formation of the reactive nitrene
is accompanied by the photo-elimination of a molecule of N2.


41
residues in the polypeptide residing near the nitrene while in
the binding site. The triplet nitrene requires a coupled
reaction before a covalent bond can form. The first reaction
can be an abstraction of an electron from a C-H bond. This
generates a short lived radical ( CH3) that can couple with
reduced nitrene to form a useful covalent bond or form some
other product. The only other major reactions are
rearrangements, to which aryl nitrenes are not susceptible
(Bayley and Knowles, 1977). The 8-Azido purine analogs do not
form tetrazoles (an isomer that forms a third five membered
nitrogen ring attached at C8 to C9 of the purine ring) in
aqueous solvents to any extent (Haley, B., personal communi
cation). These isomers of 8-Azido ATP or GTP are poor photo
reactive reagents and would quench photoaffinity reactions.
The productive covalent attachment of a ligand to a
receptor by photololysis is a competition between the rates of
photoincorporation and rates of decomposition plus
decomposition of the reagent. The rate of photoincorporation
consists of the formation of the reactive nitrene which in
turn must react with surrounding amino acid residues to insert
into heteratomic linkages such as C=0, C-S, C-N, C-H, N-H or
water. The estimates for this total reaction rate in aryl
nitrenes are relatively long, approximately 10'4sec when
measured in a soft polystyrene matrix (Reiser et al., 1968).
This suggests that if binding affinities are strong enough, at
least in the millimolar range, then the ligand should reside


42
long enough in the binding site for productive photoinduced
covalent binding to the receptor (Chowdry and Westheimer,
1979; and Potter and Haley, 1983. Additionally, the stronger
the binding affinity of the ligand for its site, fewer
residues will be available for interaction with the nitrene
increasing the likelihood of covalently labeling a specific
residue at the binding site.
Other concerns in photoaffinity labeling, besides binding
affinity as discussed above, are the temperature of incubation
and photolysis, ionic strength, pH, protein concentration,
intensity of photolyzing light and its affect upon the
receptor molecule. In the vast majority of photolabeling
experiments described in the literature, the incubation and
photolysis were done near 0C and under dim red light to
prevent decomposition of the azi do compound. This was done in
most instances to minimize the rates of exchange thereby
increasing the residency of the radiolabeled ligand in the
binding site and to dissipate heat from the irradiation by uv
light. The ionic strength and pH should be optimal to ensure
not only tight binding of ligand to the receptor but also that
photoincorporation is not diminished in some manner. In the
following study I will describe the photoincorporation of [a
and y32]8-Azido GTP in 28S ribosomes. I will show that the GTP
analog binds specifically to a single ribosomal protein and
will describe the development of optimal photolabeling
conditions for this reaction.


43
Materials and Methods
Reagents
Both [a32P]8-Azido GTP and [y32P]8-Azido GTP were obtained
as triethyl ammonium salt at specific activities of 6.7 and
10.2 Ci/mmole, respectively, from ICN radiochemicals (Irvine,
CA). GTP was obtained from Sigma Chemical Co. as a Type I
grade reagent.
Binding Assay Solutions
Reaction Buffer A: 100 mM KC1, 5 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer B: 10 mM NH4C1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer C: 100 mM KC1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Polyacrylamide Gel Solutions
Laemmli Sample Buffer: 2% SDS, 5% 2-BME, 10% glycerol, and
62.5 mM Tris-HCl, pH 6.8
ID-PAGE Tank Buffer: 24 mM Tris-HCl, pH 8.3, 92 mM Glycine,
0.2% SDS and 0.1 M sodium thioglycolate
2D-PAGE Sample Buffer: 9 M urea, 10 mM 2-BME, 60 mM
potassium acetate, pH 6.7, and 0.01% aminoethanethiol
2D-PAGE Prep. Buffer: 5 M urea, 2% SDS, and 10 mM
sodium phosphate, pH 7.2
2D-PAGE Pre-Run Buffer: 9 M urea, 57 mM potassium hydroxide,
and 340 mM HPLC grade acetic acid, pH 5.0


44
2D-PAGE Tank Buffer: 0.5% SDS, 0.1 M sodium phosphate, pH
7.2, 3 mM 2-BME and 0.1 mM thioglycolate.
rRNA Extraction Buffer: 15 mM sodium acetate, pH 5.0, 100 mM
sodium chloride, 5 mM disodium EDTA, and 2% SDS
rRNA Run Buffer: 0.5 M Tris-HCl, pH 8.3, 0.5 M boric acid,
and 10 mM EDTA.
Photolabelino of 28S Ribosomes with Radiolabeled 8-Azido~GTP
The conditions used for photoincorporation of
radiolabeled 8-Azido-GTP were nearly identical to those used
for the Mi Hi pore filter assay. The final reaction mixture
contained 28S ribosomes (0.5 and 1.0 nM for ID and 2D-PAGE
analyses, respectively) and radiolabeled NTP at varying
concentrations (0.05 to 6 nM) and GTP (5 and 10 nM for ID and
2D-PAGE analyses, respectively) if a binding site blocker was
desired. The order of addition was important. The ribosomes
and GTP (if appropriate) were mixed in the reaction buffer (A,
B, or C) and incubated on ice and in the dark for 5 min. The
radiolabeled NTP dissolved in reaction buffer was then added
to a final volume of 30 or 100 ^1 for ID or 2D-PAGE analyses,
respectively and incubated on ice in the dark for an
additional 15 min.
Photolysis was performed with a hand held ultraviolet
lamp with a purple filter that allowed transmission at 254 or
377 nm. Irradiation with 254 nm light was done in all cases
and at varying distances directly above the samples resulting
in intensities of 400 to 800 nW/cm2 for photolabeling with


45
8-Azido-GTP or GTP, respectively. The intensity of the uv
light was determined with the use of Spectroline digital uv
intensity meter (model # 254X). In initial photolabeling
experiments with [a32P]8-Azido GTP, 2500 ^W/crn2 was used.
Irradiation of samples was allowed to proceed for 5 to 10 min
typically, on ice, in the dark with only dim red light for the
8-Azido GTP and with minimal room light for GTP. Samples were
then prepared for either ID or 2D PAGE analysis.
Polyacrylamide Gel Electrophoresis
Photolabeled ribosomal samples were often resolved on ID
SDS slab gels (1.5 X 160 X 200 mm) as described by Laemmli
(1970). Samples were loaded into sample wells of a
polyacrylamide SDS gel (12% or 15%T, 3.5%C, 0.375 M Tris-HCl,
pH 8.8, and 0.1% SDS; T is the total monomer concentration, C
is the crosslinker, bisacrylamide, in percentage T) with a
stacker gel (3%T, 3.1%C). Electrophoresis was performed in
ID-PAGE tank buffer (24 mM Tris-HCl, pH 8.3, 0.192 M Glycine,
and 0.2% SDS) at an applied constant current of 10 mA for
approximately 14 h at room temperature. Following
electrophoresis the gel was fixed in 50% methanol and allowed
to equilibrate overnight with repeated changes of the
methanol. The gel was then silver stained (Wray et al.,
1981)and refixed in 50% methanol for at least 4 h to ensure
complete shrinkage of the gel before autoradiography was
performed. The stained and fixed gel was then sandwiched
between two layers of wetted Bio-gel wrap and allowed to air


46
dry while clamped in a plexigls holding frame. The dried gel
was then autoradiographed at room temperature for an
appropriate period of time.
A modified 2D-PAGE system of Leister and Dawid (1975)
was used to determine which ribosomal protein was photolabeled
by either radiolabeled 8-Azido-GTP or GTP. Following
photolabeling as described above, ribosomal samples were
extracted with urea and lithium chloride as described below.
The first dimension was run in tube gels (3 X 110 mm) cast in
siliconized (Sigmacote, Sigma) glass tubing. The acrylamide
concentration of the separation tube gel was 4.6%T, 3.2%C,
with 9 M urea, and 60 mM potassium acetate, pH 4.3. The first
dimension was pre-run in 2D-PAGE pre-run buffer (9 M urea, 60
mM potassium acetate, pH 4.3) to remove the polymerizing
agent, ammonium persulfate (0.1%). A stacker gel (4%T and
3.5%C) was then cast (3 cm long) on top of the separator tube
gel. To each nl of dialyzed sample, 0.25 nl of sample gel
(4%T, and 3.4%C) was added and then cast on top of the stacker
gel. The first dimension was then electrophoresed at a
constant amperage of 0.2 mA/gel toward the cathode in 2D-PAGE
tank buffer (35 mM B-alanine, pH 4.5, and 0.01%
aminoethanethiol) until the tracking dye, pyronine Y, had
entered the separation gel, then the current was boosted to
0.5 mA/gel. The polymerizing agent of both stacker and sample
gel was 0.1% riboflavin. When the tracker dye was within 1 cm
of the end of the tube gel, electrophoresis was stopped. The


47
tube gel was removed from the glass tube, then equilibrated in
2D-PAGE prep, buffer (5 M urea, 2% SDS, and 10 mM sodium
phosphate, pH 7.2) for 30 min while shaking gently at room
temperature.
For the second dimension a SDS slab (1.5 X 160 X 200 mm)
gel (10%T, and 3.5%C in 5.1 M urea, 0.5% SDS, 0.1 M sodium
phosphate, pH 7.2) was used. In preformed wells adjacent to
the first dimension tube gel, molecular weight markers (94,
67, 43, 30, 20.1, and 14.4 kDa) were run. The second
dimension was run at a constant current of 45 mA/gel toward
the anode in tank buffer (0.5%SDS, 0.1 M sodium phosphate, pH
7.2, and 3 mM BME).
The slab gels were fixed in 50% methanol, with several
changes, overnight. The gels were then silver stained (Wray
et al., 1981) and dried in a sandwich of Bio-gel wrap and
prepared for autoradiography.
Electrophoresis of Mitochondrial 12S rRNA
Photolabeling of mitochondrial 12S rRNA, as well as
ribosomal proteins with [a32P]8-Azido-GTP or [a32P]GTP, was
also examined. Following extraction of the photo!abeled 28S
ribosomes by urea and Li Cl, the rRNA pellet was stored at -
20!C until analyzed. The rRNA pellet was dissolved in an
extraction buffer (15 mM sodium acetate, pH 5.0, 100 mM sodium
chloride, 5 mM disodium EDTA, and 2% SDS) and extracted three
times with an equal volume (100 jil) of equilibrated phenol
followed by centrifugation at high speed in a Beckman tabletop


48
microcentrifuge for 5 min at room temperature. Sodium acetate
was then added to a final concentration of 200 mM to the
pooled aqueous phases. To precipitate the rRNA, 2.5 volumes
of ice cold 95% ethanol was added and the mixture was kept at
-20C for at least 4 h. The sample was centrifuged for 10 min
in a microcentrifuge at high speed to pellet the rRNA. The
rRNA pellet was washed two more times with 70% ethanol
followed by a final wash with 95% ethanol. The sample was
dried under vacuum for approximately 15 min.
The rRNA pellet was redissolved in a 1:1 ratio of rRNA to
formamide loading buffer (80% formamide, 10 mM EDTA, pH 8, 1
mg/ml bromophenol blue, and 1 mg/ml xylene cyanol FF) prior
to ID urea PAGE. Samples were resolved with molecular markers
of six synthetic poly(A)-tai1ed RNAs ranging in length from
0.24 to 9.49 kb (BRL, Gaithersburg, MD) on a one dimensional
urea PAGE (7.5%T, 3.5%C, 7 M urea, 0.5 M Tris-HCl, pH 8.3, 0.5
M boric acid, 10 mM EDTA, 0.05% TEMED, and 0.017% ammonium
persulfate) with a stacker gel (5%T, 3.5%C). Electrophoresis
was done in rRNA run buffer (0.5 M Tris-HCl, pH 8.3, 0.5 M
boric acid, and 10 mM EDTA) at a constant voltage of 500 V
toward the anode. The gel was then soaked in 0.2 M sodium
acetate, pH 5.0, for 30 min prior to staining with methylene
blue. The stained gel was washed with water, destained in 5%
acetic acid overnight, and then washed with water again just
prior to photography. The gel was dried overnight in a
sandwich of Bio-gel wrap and prepared for autoradiography.


49
Extraction of Ribosomal Proteins for 2D-PAGE Electrophoretic
Analvsis
Ribosomes were extracted in urea-LiCl to remove rRNA from
ribosomal proteins as described by Matthews et al. (1982)
prior to 2D-PAGE analysis. Thawed 28S ribosomes were adjusted
to 9 M urea, 3 M Li Cl with addition of solid urea and Li Cl
with a resulting neutral pH. The samples were stirred
overnight at 4C followed by centrifugation at 50,000 rpm
(100,000 X g) for 60 min in a Beckman type 65 rotor to
precipitate the rRNA. The supernatant containing ribosomal
proteins was removed and the rRNA pellet was extracted once
again, by stirring for 2 h at 4C, with the above buffer.
After centrifugation the supernatants were pooled and dialyzed
against 2D-PAGE sample buffer (9 M urea, 0.01%
aminoethanethiol, 10 mM BME, and 60 mM potassium acetate, pH
6.7) overnight at room temperature in preparation for 2D-PAGE
analysis. The rRNA pellets were stored in the type 65
centrifuge tubes at -20C until needed.
Results
fg32Pl8~Azido GTP Photolabels a 38 kDa Ribosomal Protein in a
Specific Manner.
Filter binding studies suggest that a single and
specific binding site for GTP exists on the mitochondrial
ribosomal small subunit. To identify which ribosomal
protein(s) may be involved a photochemical approach was used.
28S ribosomes were incubated with increasing concentrations
(0.15 to 6.0 nM) of [a32P]8-Azido GTP in reaction buffer B.


50
The samples were irradiated with uv (254 nm) light for 20 min
at 2500 nW/cm2 on ice (and in the dark). The photoaffinity
labeled ribosomes were resolved on a 12% polyacrylamide
Laemmli gel, silver stained and prepared for autoradiography
as described in Methods. Photoaffinity labeling of ribosomal
small subunits with [a32P] 8-Azido GTP yielded a single
specifically radiolabeled 38 kDa ribosomal protein (figure 3-
2A). The addition of excess unlabeled GTP blocked
photolabeling of this protein, suggesting that the binding
site was also specific for GTP (figure 3-2 A). The amount of
radiolabeling increased as the concentration of the [a32P]8-
Azido GTP increased but is approaching saturation at 6^M
[a32P]8-Azido GTP. This is consistent with the apparent Kd of
1.92 jiM (75% of the binding sites on 0.5^M 28S ribosomes
should be occupied at this Kd and concentration of 8-Azido GTP
used in this experiment) determined by filter binding assays
(table 2-3). Several ribosomal proteins with Mr > 40 kDa were
nonspecifical ly photolabeled at concentrations of [a32P]8-Azido
GTP above the Kd for 8-Azido GTP.
Unambiguous identification of the specifically photo-
labeled 40 kDa ribosomal protein was not possible by one
dimensional gel analysis due to the existence of other 28S
ribosomal proteins of similar molecular weight. Two dimen
sional gel analysis of photoaffinity labeled small subunits
was done in order to do so. In figure 3-3 a single ribosomal
protein, S5, was specifically photolabeled with [cc32P]8-Azido


51
8N3GTP(/iM) 0.3
GTP (5/iM) +
0.15 0.3 0.8 1.5 6.0
-+- + + + +
Figure 3-2 A Photoaffinity labeling of 28S ribosomes with
[a P]8-Azido GTP. Bovine 28S ribosomes (0.5 nM) were
irradiated in the presence of increasing concentrations (0.15
to 6 *iM) of the 8-azido analog of GTP in reaction buffer B.
The samples were irradiated with uv light for 20 min at 2500
nW/cm on ice under dim red light. The specific activity of
the [a P]8-Azido GTP was 1.35 Ci/mmol just prior to use. In
panel A a representative lane of silver stained (S) proteins
electrophoresed on a 12% SDS-polyacrylamide gel and the
corresponding autoradiogram (AR) are shown. In alternate
assays the GTP binding site was blocked by the addition (+) of
5uM GTP. The Mr markers were as follows: Phosphorylase b (94
kDa), Bovine Serum Albumin (67 kDa), Ovalbumin (43 kDa),
Carbonic Anhydrase (30 kDa), Soybean Trypsin Inhibitor (20.1
kDa), and alpha-Lactalbumin (14.4 kDa). these same Mr markers
were used throughout this entire study. The arrow indicates
the presence of a 38 kDa protein specifically radiolabeled
with [a P]8~Azido GTP. See Methods for additional details.


52
x
o
LlI
X
¡2
CL
Id
>
5
Id
a:
8N3GTP CONCENTRATION (X 106M)
Figure 3-2 B Densitometric analysis of the autoradiogram for
the specifically radiolabeled 38 kDa ribosomal protein in
figure 3-2 A is shown. Data were the relative peak height
densi tometri c measurements of the 38 kDa band (panel A) in the
presence of increasing concentrations of [a P]8-Azido GTP and
the absence of GTP.


53
A
B
Figure 3-3 Two dimensional PAGE analysis of photoaffinity labeled
28b ribosomes with [a P]8-Azido GTP. Bovine Z8S ribosomes (1 ^M)
were irradiated in the presence of 2 *iM [a P]8-Azido GTP (45%
saturation) and in the presence (B) and absence (A) of 10 ^M GTP.
The samples were irradiated with uv light for 10 min at 2500 ^W/cm
on ice as described in Methods. The specific activity of the
[a P]8-Azido GTP was 1 Ci/mmol just prior to use. Silver stained
2D-PAGE gels are on the left and their corresponding autoradiograms
are on the right. Autoradiograms were exposed for 14 days. The
molecular weight markers used were the same ones described in
figure 3-2. The arrows indicate the presence of the nonspecific-
ally labeled (S4) and the specifically labeled (S5) proteins. See
Methods for additional experimental details.


54
GTP. The presence of excess amounts of unlabeled GTP com
pletely blocked photoincorporation of [a32P] 8-Azido GTP into
S5. Another ribosomal protein, S4, was also labeled
nonspecifically, since the presence of excess amounts of GTP
did not block photolabeling of S4.
Development of Optimal Photoaffinitv Labeling Conditions for
8-Azido GTP
The extent of specific photolabeling of S5 by [a32P]8-
Azido GTP was small, especially in comparison to the much
greater nonspecific labeling of S4 (figure 3-3). Optimization
of photoirradiation and ionic conditions was undertaken to
improve the quality of the photoaffinity labeling of 28S
ribosomes by 8-Azido GTP. Optimal conditions were determined
by both filter binding assays and photoaffinity labeling
methods.
The development of optimal ionic conditions focused on
the modification of two components of the reaction buffer B as
developed by Bodley (Bacca, et al., 1976). Bodley developed
this reaction buffer for the study of GTP hydrolysis by EFG in
bacterial systems. One component, NH4C1, is known to be toxic
to peptidyl transferase activity (Denslow and O'Brien,
personal communication) on the large subunit of the
mitochondrial ribosome and its replacement with another
monovalent salt such as KC1 was studied.
In table 3-1 the effect of replacing 10 mM NH4C1 with KC1
on the binding of [8-3H]GTP to 28S ribosomes was examined by


55
filter binding assays. Increasing concentrations of [a32P]GTP
were incubated with 0.1 jiM 28S ribosomes in reaction buffer B
(treatment #1 in table 3-1) as a control, and the test
reactions were in the same reaction buffer but with 10 mM
NH4C1 replaced with increasing concentrations (10 to 100 mM)
of KC1 and decreasing concentrations of MgCl2. The
substitution of NH4C1 by KC1 or the reduction of MgCl2 from 20
to 5 mM did not alter the binding of [a32P]GTP to 28S
ribosomes. The most stringent condition, 100 mM KC1 and 20 mM
MgCl2, supports binding of [8-3H]GTP to the 28S ribosome in an
equivalent manner as controls. This reaction buffer C (100 mM
KC1, 20 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME) and
was used in experiments discussed below.
Potter and Haley (1983) reported that in their
experience with a number of azido-purine analogs, photolysis
at 254 nm and at intensities of 180 to 800 jiW/cm2 for 1 to 10
min caused little if any detectable damage in a variety of
enzymatic systems. In our initial experiments (figure 3-2 and
3-3) these guidelines were exceeded.
The intensity and duration of photoirradiation was
reduced to evaluate the minimal amount of uv irradiation
necessary to adequately photolabel 28S ribosomes with [a32P]8-
Azido GTP. Ribosomes (0.5 nM) were photolabeled in the
presence of 0.75 nM [ approximately 24% saturation of the GTP binding site
(calculation based upon a Kd of 1.92 nM) in reaction buffer B.


56
Table 3-1
The Effect of Substituting KC1 for NfyCL and the Lowering of
the MgCl, Concentration in the Reaction Buffer on the
Noncovalent Binding of la PI GTP to 28$ Ribosomes
eatment
#
NH,C1
fmM)
MgCl,
(mM)
KC1
(mM)
a
fmM)
Kd
SE
(nM)
n

SE
1 (B)
10
20
75
3.8

2.2
0.91

0.08
2
-
20
10
75
4.0

1.0
0.99

0.05
3
-
20
20
85
3.1

0.8
0.92

0.04
4
-
20
40
105
1.7

1.0
0.96

0.11
5
-
20
60
125
8.3

3.4
0.91

0.07
6 (C)
-
20
100
165
5.8

2.5
0.87

0.06
7
-
10
100
135
2.5

0.9
0.92

0.06
8 (A)
-
5
100
120
2.7

0.8
1.00

0.06
Scatchard analysis for the binding of [ ribosomes under various ionic conditions by tne Mi 11ipore
filter assay. The data are binding affinities (Kd) and number
of binding sites (n) for a single binding curve. The
concentration of the ribosomes was 0.1 nM and the JV P]GTP
0.02 to 1 liM. The specific activity of the [a PJGTP was
diluted to 5.4 Ci/mmol with GTP just prior to use. Reaction
buffers A, B, and C (for complete formulations see Methods)
are anotated in parentheses, beside their respective
treatments In all reaction buffers ImM BME was used. No
differences were observed with the substitution of NH4C1 in
reaction buffer B (treatment #1) for increasing concentrations
of KC1 (treatments 2 to 6) or decreasing concentrations of
MgCl2 (treatments 7 & 8).
an = total ionic strength
In figure 3-4 A the reduction of 254 nm uv irradiation from
2500 to 800 nW/cm2 for 10 min reduced the photolabeling of the
38 kDa ribosomal protein (no excess GTP present) by only 47%,
as determined by densitometric analysis (figure 3-4 B). The
amount of labeling was still adequate for analysis. However,
irradiation for 2 min reduced photolabeling of the same 38 kDa
protein by 75 to 90% at 2500 and 800 nW/cm2, respectively.


57
This provided minimal detectable labeling at 2500 ^W/crn2 and
virtually no labeling at the lower level of uv irradiation
even after a 2 week exposure of the autoradiogram. The
pattern of photolabeling remained the same as seen in figure
3-2 A. Photolabeling of the 38 kDa protein by [a32P]8-Azido
GTP was blocked in the presence of excess GTP and not in the
case of the 43 kDa ribosomal protein.
In an continuing effort to find the minimal exposure of
ribosomes with uv light for adequate photolabeling [y32P]8-
Azido GTP was used. This isotope was used because [a32P]8-
Azido GTP was no longer available. [y32P]8-Azido GTP was also
available in higher purity (>98%), more than twice the
specific activity (10.2 Ci/mmole),and at lower cost. Optimal
photoirradiation conditions for the photolabeling of 28S
ribosomes with [y32P]8-Azido GTP were determined in reaction
buffer C (100 mM KC1, 20 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and
1 mM BME). Substoichiometric concentrations of [y32P]8-Azido
GTP (0.43 *iM), corresponding to approximately 18% binding,
were incubated with 28S ribosomes (0.5 ^M) and irradiated at
300 and 800 ^W/cm2 for 4 and 8 min on ice in the dark. The
minimal uv (254 nm) irradiation intensity and duration found
for adequate labeling were 300 nW/cm2 for 10 min (figure 3-5
A and B). These conditions were utilized in subsequent
studies involving this analog of GTP.


58
Ribosomes
GTP (5/iM)
Irrad. time (min)
-++ + + + + + +
+ + + +
10 2 10 2
Intensity (/iW/cm^) 2500
800
Figure 3-4 A Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S ribosomes with
[ P]8-Azido GTP. 28S ribosomes (0.5 uM) were incubated with
0.75 liM [a P]8-Azido GTP (24% saturation) in reaction buffer
B. The specific activity of the [a P]8-Azido GTP was 1.0
Ci/mmol. In panel A is a representative lane of silver
stained ribosomal proteins on the left and the corresponding
autoradiogram on the right. The AR was exposed for 14 days
before development. Tne presence (+) and absence (-) of
ribosomes and unlabeled GTP as well as the intensity and
duration of uv (254 nm) irradiation are noted below the
autoradiogram. The arrow indicates the position of the
specifically radiolabeled 38 kDa protein. For more
experimental details see Methods.


RELATIVE PEAK HEIGHT
59
[a32P]8-Az¡do GTP
800/xW / cm2 2500/xW / cm2
Figure 3-4 B Densitometric analysis of the autoradiogram in
figure 3-4 A is shown. Densitometric analysis was done for
the specifically radiolabeled ribosomal protein (38 kDa) and
the nonspecifical ly labeled protein (43 kDa) ribosomal protein
at varying intensities and duration of uv irradiation. Note
that in tne presence (+) of 5 p.M GTP the 38 kDa protein was
not radiolabeled. For additional experimental details see
Methods.


60
GTP (5/i.M ) -+-+-+-+
Irrad.time (min) 4 10 4 10
Intensity (^W/cm^) 300 800
Figure 3-5 A Continued optimization of the intensity and
duration of uv irradiation for photoaffinity labeling of 28S
ribosomes with [y P]8-Azido GTP. 28S ribosomes (0.5 jiM) were
incubated with 0.5 nM [y P]8-Azido GTP (18% saturation) in
reaction buffer C. The specific activity of the [y P]8-Azido
GTP was 8.4 Ci/mmol. In panel A is a representative lane of
silver stained (S) ribosomal proteins on the left and the
corresponding autoradiogram (AR) on the right. The AR was
exposed for 3 days before development. The presence (+) and
absence (-) of unlabeled GTP as well as the intensity and
duration of uv (254 nm) irradiation are noted below the
autoradiogram.


61
032P]8-Az¡do GTP
x
o
J
X
¡5
CL
Ld
>
5
UJ
cc
300/W / cm2
800/W / cm2
Figure 3-5 B Densitometric analysis of the autoradiogram in
figure 3-5 A is shown. Densitometric analysis was done for
the specifically labeled ribosomal protein (38 kDa) and the
nonspecifically labeled protein (43 kDa) ribosomal protein at
varying intensities and duration of uv irradiation. The
presence (+) and absence (-) of 5 nM GTP is indicated below
the figure. In the presence of excess amounts of GTP (+) the
38 kDa protein was not radiolabeled. For additional
experimental details see Methods.


62
No significant differences were seen in the photolabeling
pattern of the two isotopes ( compare figures 3-4 A and 3-5 A)
though the intensity of radiolabeling was greater for the
[y32P] isotope due to its higher specific activity. The
[y32P]8-Azido GTP was utilized in the remainder of 8-Azido GTP
photoaffinity experiments described in this paper.
The last component of the reaction buffer to be modified
was the divalent cation, MgCl2. MgCl2 has been shown by
Denslow et al. (1988) to promote adherence of 28 and 39S
subunits to one another due presumably to salt bridging
effects at higher concentrations of the divalent cation. To
reduce the possibility of nonspecific binding due to salt
bridging of the nucleotide, MgCl2 concentration was reduced
from 20 to 5 mM and photolabeling was examined under these
conditions. The same incubation conditions were used as
described in the above experiment and uv photoirradiation was
done at 300 ^W/cm2 for 10 min. The 38 kDa ribosomal protein
was still specifically photolabeled (figure 3-6 A) by the GTP
analog, [y32P]8-Azido GTP, and a 45% increase in this labeling
was observed by densitometric analysis as the concentration of
MgCl2 dropped (figure 3-6 B) to 5 mM. Since the lowest
concentration of MgCl2 provided a modest increase in
photolabeling of the specifically labeled 38 kDa ribosomal
protein it was adopted for subsequent studies. The amount
oflabeling on the nonspecifically labeled 43 kDa ribosomal
protein was unchanged in the absence of excess GTP but did
increase 47% in the presence of 5 mM GTP.


63
s
AR
94
67
43
fi
30
-1
20.1
X
14.4
1
G TP(5/iM)
-+-+- +
MgCL2(mM)
20 10 5
0.5 uM
uv light
activity of the
Figure 3-6 A The effect of decreasing concentrations of MgCl2
on photoaffinity labeling of 28S ribosomes with [y P]8-Azido
GTP. Bovine 28S ribosomes (0.5 uM) were incubated witn
y P]8-Azido GTP (18% saturation) and irradiated with
;or 10 min at 300 ^W/cm on ice. The specific ac
[y P]8-Azido GTP was 6.3 Ci/mmol. The concentration of the
MgCl2 component of the reaction buffer was decreased from 20
mM (reaction buffer C) to 5 mM (reaction buffer A). In panel
A a representative silver stained lane of 28S ribosomal
proteins and molecular weight markers are shown with the
corresponding autoradiogram on the right. The concentration
of the MgCl2 component of the reaction buffer and the presence
(+) and absence (-) of 5 nM unlabeled GTP is shown below the
autoradiogram. The autoradiogram was exposed for 3.5 days
before developing. The arrow indicates the position of the 38
kDa specifically radiolabeled protein.


64
t-
x
CD
UJ
X

X
UJ
>
5
L
O'
Figure 3-6 B Densitometric analysis of the autoradiogram in
figure 3-6 A. The amount of radiolabeling of the 38 and 43
kDa ribosomal proteins was plotted versus the concentration of
MgClp in the reaction buffer. Note that no radiolabeling of
the 38 kDa protein was detected in the presence of 5 GTP.


65
The three previous experiments suggest that the optimal
ionic composition of the reaction buffer for the binding of
GTP and its 8-Azido analog to 28S ribosomes was 100 mM KC1, 5
mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME and will be
referred to as reaction buffer A in this paper. The optimal
uv photolabeling conditions were 300 jiW/cm2 for 10 min for 8-
Azido GTP.
With these new conditions another attempt was made to
analyze photolabeled ribosomal proteins by two dimensional
PAGE. In figure 3-7 A to F, 28S ribosomes (1 ^M) were
photoaffinity labeled with increasing concentrations of
[y32P]8-Azido GTP (0.3, 1, and 3 ^M) in the presence and
absence of excess (10 |iM) GTP. These amounts of [y32P]8-Azido
GTP correspond to 9, 20, and 60% saturation of the GTP binding
site on the ribosome (based on a Kd of 1.92 tiM). Small
subunit ribosomes were resolved by 2D-PAGE and radiolabeled
proteins were detected by autoradiography.
At all concentrations tested, S5 was specifically
radiolabeled by [y32P]8-Azido GTP (figure 3-7 A to F) and the
radiolabeling increased directly proportional to the
concentration of [y32P]8-Azido GTP (figure 3-7 G). The
presence of GTP completely blocked photoincorporation of the
GTP analog into S5. S4 was also radiolabeled, but in a
nonspecific manner, since excess amounts of GTP failed to
block photoincorporation of [y32P]8-Azido GTP. These results
support the tentative finding seen in the initial attempt to


Figure 3-7 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with increasing concentrations of
[y P]8-Azido GTP. Bovine 28S ribosomes (l pM) were irradiated
with uv light for 10 min at 300 nW/cm on ice in the presence
of increasing concentrations of [v Pl8-Azido GTP (0.3 to 3
Results in the presence (B, D, & F) and absence (A, C,
of 10 nM GTP is also shown. The samples were irradiated
witn uv light for 10 min at 300 ^W/cm nice and under dim
red light. The specific activity of the [y P]8-Azido GTP was
2.9 Ci/mmol just prior to use. Silver stained gels are on the
left and their corresponding autoradiograms on the right.
Autoradiograms were exposed for 7 days. The Mr markers used
were the same ones described in figure 3-2. See Methods for
additional experimental details.
i tn


67
Figure 3-7 A & B Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with 0.3 nM (9% saturation) [y P]8-Azido GTP
(panels A & B). The presence of 10 nM GTP (panel B) blocked
radiolabeling of S5.


68
Figure 3-7 C & D Two dimensional PAGE analysis of_ohotoaffinity
labeled 28S ribosomes with 1.0 nM (28% saturation) [y P]8-Azido GTP
(panels C & D). Note, that in the presence of 10 GTP (panel D)
$5 was not radiolabeled.


69
Figure 3-7 E & F Two dimensional PAGE analysis of Dhotnaff-ini+v
(panefs E8& Fl^Not! W1thht3*-0 saturation! [Y32P]8-Azido G{>
5 wfs not radiolabeled preSence of mM dp (panel F)


70
[r32p]8N3GTP CONCENTRATION (X106M)
Figure 3-7 G Densitometric analysis of the GTP specific and
nonspecific radiolabeling of ribosomal proteins S5 and S4
respectively. In panel G densitometric analysis of the
autoradiograms in panels A to F for the specific labeling of
S5 and nonspecific labeling of S4 is shown.


71
GTP(lOjuM) + +
Figure 3-7 H Analysis of rRNA extracted from 28S ribosomes
irradiated in the presence of [y P]8-Azido GTP. This is an
analysis of the rRNA extracted from photoaffinity labeled 28S
ribosomes shown in figure 3-8, panels C & D ( the result in
all other panels was the same, no radiolabeling of rRNA). The
rRNA was extracted following uv irradiation by 9M urea and 3M
Li Cl and el ectrophoresed by ID urea PAGE (7.5% PAGE) as
described in Methods. The left panel is a methylene blue
stained gel (S) and its corresponding autoradiogram (AR) on
the right. The autoradiogram was exposed for 41 days. The
molecular weight markers are six synthetic poly(A)-tailed RNAs
ranging in length from 0.24 to 9.49 kb (Bethesda Research
Labs). No rRNA fragments were radiolabeled.


72
identify the GTP binding protein by 2D-PAGE analysis (figure
3-3), that S5 was specifically labeled by 8-Azido GTP. The
development of more stringent ionic and milder photoirradia
tion conditions did not eliminate nonspecific labeling of S4.
S4 was the most intensely labeled protein which increased
proportionally with the concentration of [y32P]8-Azido GTP.
S4 appears to be reciprocally labeled in the presence of
excess amounts of GTP (figure 3-7 G). The amount of S4
labeling increases in the presence of 10 *iM GTP, even though
the radionucleotide was diluted as much as 33 fold. This
suggests this site may be a lower affinity site for 8-Azido
GTP. No radiolabeling of 12S rRNA was detected (figure 3-7
H), suggesting no direct involvement of rRNA in binding of 8-
Azido GTP to 28S ribosomes.
Piscussion
Photoaffinity labeling of 28S ribosomes with 8-Azido GTP
revealed that a single 38 kDa ribosomal protein bound the GTP
analog in a specific manner (figure 3-2 to 3-7). Unlabeled
GTP could displace the radiolabeling of this protein
suggesting the site was specific for GTP. A 43 kDa protein
was also radiolabeled but the labeling was nonspecific because
excess amounts of GTP failed to block binding. The amount of
radiolabeling on the 38 kDa protein increased with increasing
concentrations of [a32P]8-Azido GTP and was saturable (figure
3-2 B) in a fashion consistent with the binding of this
compound to 28S ribosomes as determined by filter binding


73
assays (figure 2-3). The photolabeling reaction was done
under the same ionic conditions (reaction buffer B) as were
used in filter binding assays to assess [a32P]8-Azido GTP
binding to 28S ribosomes.
The identity of this labeled protein was examined by 2-
dimensional PAGE. Two proteins retained the label, one S4,
was the predominately radiolabeled protein but the labeling
was nonspecific because it was not diminished in the presence
of excess amounts of GTP. The second protein, S5, was
specifically radiolabeled by [a32P]8-Azido GTP. Excess
concentrations of GTP blocked binding to this protein
suggesting that it is the specific binding site for GTP.
Comparing these results to those of the 1-dimensional
analysis (figure 3-2), the 43 kDa nonspecifical ly labeled
protein correlates to S4 in both Mr and binding specificity,
and the 38 kDa protein would be S5 for the same reasons. The
only disparity was that the radiolabeling of S5 was less than
that seen on S4 in the two-dimensional PAGE analysis (figure
3-7). The disparity likely resides in differences in sample
preparation. In two-dimensional PAGE analysis, photolabeled
28S ribosomes are extracted under acidic conditions (pH 2.5)
to remove rRNA prior to separation in the first dimension in
urea also under acidic conditions (pH 4.3). Temple et al.
(1966) and Maliarik and Goldstein (1988) have observed that
some covalent bonds formed by the aryl (purine) nitrene
adducts are acid labile. In previous attempts to resolve


74
photolabeled ribosomes with the azido analog no radiolabeled
proteins were detected. Increasing the pH of the extraction
step to neutrality, only one radiolabeled protein, S4, was
found and it was not blocked in the presence of GTP (data not
shown). However when the pH of the first dimensional
separation was raised from 4.3 to 5.0 we were able to observe
not only the nonspecific labeling of S4 but also specific
labeling on S5 (figure 3-3 and 3-7). The separation of these
basic ribosomal proteins in the first dimension in urea could
not be done at any higher pH without risking alteration of the
relative mobilities of the proteins in a defined system
(Matthews et al.,1982). Never-the-less, under the elevated pH
conditions during sample preparation and analysis, it is clear
that S5 is the protein binding GTP in a specific manner.
Concerns about the survivability of the covalent bound formed
by the nitrene adduct, and photolabeling under more
physiological ionic conditions lead to studies to determine
the minimum photoirradiation required and optimal ionic
conditions for this reaction.
The minimum fluency (pW/cm2) and duration of irradiation
to achieve labeling of ribosomal proteins using 254 nm light
with 8-Azido GTP, were 300 ^W/cm2 and 10 min, respectively
(figure 3-5). This was determined over the span of two
experiments in which both the [a32P] and [y32P]8-Azido GTP were
used, with no differences seen in the pattern of radiolabeled
proteins. O'Brien et al. (personal communication) found that


75
binding of [y32P]GTP to 28S ribosomes, under conditions similar
to those used here, resulted in no hydrolysis of the [y32P]
label even after several hours of incubation with the
ribosome. This indicates that under these binding conditions
[y32P]8-Azido GTP can be used with no hydrolysis of the [y32P]
to obscure the results of photoaffinity labeling.
The length of time required (10 min) for adequate levels
of radiolabeling are somewhat longer than reported by others
(typically 2 to 6 min at fluency < 1000 nW/cm2 on ice in
various buffer biological solutions, see Potter and Haley,
1983). However, irradiation for 4 min at 300 W/cm2 was
minimally acceptable in 1-dimensional systems, but due to
greater concentrations of protein (1 versus 0.5^M ribosomes)
it was not judged adequate for 2-dimensional analysis.
Several enzymatic systems which were subjected to
photoaffinity labeling saw no appreciable loss in function
when irradiated with 254 nm light at low temperatures for as
long as 10 min if the fluency remained below 800 W/cm2 (Potter
and Haley, 1983). These guidelines were used, since no
functional assay exist for the 28S ribosome which could be
used to monitor uv induced damage.
Geahlen and Haley (1977) reported that the half-life of
the 8-azido GMP (160 nM at neutral pH) was approximately 20
sec in protein free buffer solution at approximately 1000
nW/cm2 with 254 nm light. Since only minute quantities of the
8-azido GTP were available, it was not possible to examine the


76
uv degradation of the 8-azido to N2 and 8-NH2-GTP by
spectrophotometric means as done by Geahlen and Haley (1977).
Also, no suitable thin layer chromatography or other method
could be found that could discriminate between 8-azido and the
8-NH2 GTP though it was determined that the gamma phosphate
was not lost following irradiation under the conditions used
in this study (data not shown).
Efforts to develop more physiologically relevant ionic
conditions for GTP binding to 28S ribosomes focused upon two
components of the reaction buffer, MgCl2, and NH4C1. In the
initial binding experiments of this study the ionic
composition of the reaction buffer (B) was adopted from the
work of Bodley (Bacca, et al., 1976) who developed it to study
GTP binding to bacterial EF-G. The concentration of the
divalent cation, MgCl2 (20 mM), was reduced to 5 mM to
alleviate concerns about subunit dimerization which could
alter GTP binding to the ribosome. O'Brien (1971) showed that
the mitochondrial monoribosomes are stabilized at the higher
concentration of MgCl2 (20 mM MgCl2 in 100 mM KC1) or greater.
This occurs because divalent cations form salt bridges which
stabilize rRNA interactions between subunits which results in
subunit association or aggregation. At 5 mM MgCl2,
mitoribosomes subunits exist as discrete particles. GTP-Mg+2
binds as a complex to GTP-binding proteins. The Mg+2 ion binds
tightly and nM concentrations of the divalent cation commonly
suffice to fulfill this requirement (Gilman, 1987). Though


77
only modest increases in the photoincorporation of [y32P]8-
Azido GTP in 28S ribosomes were observed at 5 mM MgCl2
(figure 3-6), this reduced concentration of the divalent
cation was adopted.
The replacement of the monovalent cation NH4+ by K+ was
examined because NH4C1 is known to be toxic to the peptidyl
transferase activity of the mitoribosome large subunit
(Denslow and OBrien, personal communication). Though we are
studying a nucleotide binding activity on the small subunit it
was deemed prudent to replace this monovalent cation in the
event NH4C1 is also toxic to some function of the small
subunit, and in the event that future studies of peptidyl
transferase activity might be undertaken in conjunction with
this GTP-binding activity. Additionally, we wanted to
increase the ionic strength to determine if GTP-binding would
be affected by increasing the stringency to more
physiologically relevant conditions. This was accomplished by
replacing NH4C1 with KC1 and testing the effect of higher
concentrations of KC1, equivalent to physiological
concentrations (70 to 100 mM). GTP-binding activity was
essentially unaltered at 100 mM KC1 when compared to binding
activity at 10 mM NH4C1 (table 3-1). This indicated that GTP
can bind the 28S ribosome even at moderate salt concentrations
suggesting the binding is not predominately electrostatic in
nature. Having established new ionic conditions (reaction
buffer A) photoaffinity labeling of 28S ribosomes by


78
[y32P]8-Azido GTP was examined again. Photoaffinity labeling
with increasing concentrations of 8-Azido GTP to a fixed
concentra-tion of the ribosome, resulted in radiolabeling of
the same proteins seen previously. S4 was nonspecifically
radiolabeled and S5 was specifically radiolabeled by [y32P]8-
Azido GTP. At very high saturating concentrations of the GTP
analog, other ribosomal proteins were also labeled in a
nonspecific manner (figure 3-7 E). At all concentrations of
[y32P] 8-Azi do GTP used, S5 radiolabeling was blocked in the
presence of excess GTP and the radiolabeling of S5 increased
with increased concentrations of the [y32P]8-Azido GTP.
Nonspecific radio-labeling of S4 also increased in a manner
comparable to that seen for S5 but interestingly S4
radiolabeling increased in the presence of excess GTP and
appeared to be saturable (figure 3-7 G). This suggested that
S4 bound some of the [y32P] 8-Azi do GTP displaced from the
binding site by GTP (10 nM) even though the specific activity
of the radiolabeled GTP analog was decreased by 33 to 3.3
fold. This suggests that S4 binds GTP in a manner different
than for S5, and is likely to have a lower affinity binding
site.


CHAPTER 4
PHOTOAFFINITY LABELING OF 28S RIBOSOMES BY [a32P]GTP
Introduction
Direct photoaffinity labeling of phosphofructokinase
(Ferguson, and Maclnnes, 1980) and tubulin (Nath, et al.,
1985; Hesse, et al., 1987;and Linse and Mandelkow, 1988) with
radiolabeled cAMP and GTP/GDP, respectively, encouraged us to
explore the use of the [a32P]GTP to photolabel the 28S ribo
some. The chemical structure at the site of covalent attach
ment for these purines is not known, but the fact that maximal
photolabeling of phosphofructokinase occurred at a wavelength
equivalent to its absorption maxima suggests that the adenine
portion of the nucleotide is the light sensitive chromophore
(Ferguson and Maclnnes, 1980). The use of the natural ligand
takes advantage of the increased binding affinity thereby
circumventing difficulties encountered due to lowered binding
affinities of the 8-Azido GTP analog for the 28S ribosome
(table 2-3). The disadvantages are that the mechanism of
photoactivation is unknown and therefore we do not know if
covalent bond formation is as nonselective a process as was
the case for the nitrene. Another disadvantage is that the
minimal quantum yield of product would be lower than for the
79


80
nitrene. In the case of cAMP, photolabeling of phosphofructo-
kinase was reported to be approximately 0.01% (Ferguson and
Maclnnes, 1980) several hundred times less than for the
nitrene. However that difficulty can be overcome to some
degree because [a32P]GTP can be obtained at specific activities
as much as 300 to 600 times greater than [a32P]- or [Y32P]8-
Azido GTP, respectively.
In this study we report successful photoaffinity labeling
of 28S ribosomes with [a32P]GTP. The photolabeling of ribo-
somal proteins was equivalent to that seen for [a32P]- and
[Y32P]8-Azido GTP, confirming that S5 is the specific site for
photoincorporation of GTP.
Materials and Methods
Reagents
[a32P]GTP, and ATP and [Y32P]ATP in triethylammoniurn salt
were purchased from Amersham at specific activities of 410 and
>3000 Ci/mmole, respectively. All unlabeled nucleotides were
purchased from Sigma Chemical Co. as Type I reagents as
described above.
Solutions
The same solutions as described above, in 8-Azido GTP
photoaffinity labeling experiments, were used in these
studies.


81
Methods
The same methods as described above, in 8-Azido GTP
photoaffinity labeling experiments, were used in these
studies.
Results
Photoaffinitv Labeling of 28S Ribosomes with lV2PlGTP
In an effort to substantiate the above findings
photolabeling of small subunits with [a32P]GTP was attempted.
Hesse et al. (1987) successfully photolabeled fi-tubulin with
[o32P]GTP suggesting the natural ligand could be used to
photoaffinity label the GTP binding ribosomal protein.
First, photolabeling conditions were varied to optimize
photolabeling. 28S Ribosomes (0.5 *iM) were photoaffinity
labeled in presence of 0.5 nM [a32P]GTP, which corresponds to
80% saturation (calculation based on a Kd of 15.2 nM) of the
GTP binding site. Ribosomes were photolabeled for 2 and 10
min at increasing intensities (800, 1500, 2000, and 2500
nW/cm2) of uv light (254 nm) on ice and under dim room light.
The pattern of radiolabeling of 28S ribosomes by [a32P]GTP
(figure 4-1) was similar to that for [a32P]8-Azido GTP (figure
3-4) and [y32P]8-Azido GTP (figure 3-5). The 38 kDa ribosomal
protein was specifically labeled by GTP because excess amounts
of GTP blocked labeling of that protein. The 43 kDa ribosomal
protein was still nonspecifically labeled in the presence of
excess amounts of GTP as seen with the 8-Azido analog.


82
Ribosomes
+ + + + + + + + + +-
G TP (5/iM)
Irrad. time (min) 2 10 2 2 2 5 10 200
Intensity (mW/cm2) 0.8 1.5 2.01 2.5 1 0
Figure 4-1 A Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S ribosomes with
GTP. 28S ribosomes (0.5
8-Azido GTP (80% saturation,
es
were
i n
M
incubated with 0.5
reaction buffer B. The
were irradiated with uv light for 5 min at 800 nW/cm ,
on ice, prior to electrophoresis. [a PlGTP was diluted 10
fold with GTP to a specific activity of 32 Ci/mmol just prior
to use. In panel A is a representative lane of silver stained
(S) ribosomal proteins on the right and the corresponds
autoradiogram (AR) on the left. Tne AR was exposed for 24
before development. The presence and absence of ribosomes and
unlabeled GTP as well as the intensity and duration of uv (254
nm) irradiation are noted below the autoradiogram. The arrow
indicates the radiolabeled 38 kDa protein that is specifically
labeled by GTP.


83
[a32P] GTP
x
o
UJ
X
¡2
CL
UJ
>
UJ
m
0.8mW 1.5mW 2.0mW 2.5mW
Figure 4-1 B Densitometric analysis of the autoradiogram
shown in figure 4-1 A. Densitometric analysis was done for
the specifically labeled ribosomal protein (38 kDa) and the
nonspecifically labeled protein (43 kDa) ribosomal protein at
varying intensities and duration of uv irradiation. In the
presence (+) of 5 nM GTP, the 38 kDa protein was not
radiolabeled. The duration and intensity of uv irradiation is
indicated below the figure. For additional experimental
details see Methods.


Full Text
CHARACTERIZATION AND IDENTIFICATION OF A NOVEL GUANINE
NUCLEOTIDE BINDING SITE ON THE BOVINE MITOCHONDRIAL RIBOSOME
by
JOHN CLAUDE ANDERS
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
1990

ACKNOWLEDGEMENTS
This work was made possible first by the love and support
of my wife, Valerie, and children, Jane, Benjamin, and
Katherine. Secondly, thanks are given to the U.S. Army which
provided the financial support for my graduate training. I
must thank Dr. Thomas W. O'Brien for his mentorship as my
major professor and the helpful guidance of the members of my
graduate studies committee. A special relationship often
develops between a student and their major professor and this
has been the case for me. Special thanks go to Dr. Nancy D.
Denslow who was instrumental in the initial discovery of GTP
binding to mitochondrial 28S ribosomes and also provided free
access to her work pertinent to this study and very helpful
insight and guidance. I must thank all the members of the
O'Brien laboratory for their patience in answering my
innumerable questions and especially to Mr. Michael Bryant who
often lead me through new techniques. Thanks go to fellow
graduate student Scott E. Fiesler, who collaborated with me in
the study of [a32P]ATP binding to mitochondrial ribosomes
presented in this study. Finally, thanks go to Michael Bryant
again, and to Mr. Bennie Parten of the protein core facility
in assistance with the chemical sequencing of mitochondrial
ribosomal proteins and peptides.

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABBREVIATIONS ix
ABSTRACT xv
CHAPTERS
1 INTRODUCTION 1
An Overview of GTP-Binding Proteins 1
Protein Synthesis 3
Conserved Amino Acid Sequences Among GTP-Binding
Proteins 6
Proteins Synthesis in Mitochondria 12
Proposal 16
2 NONCOVALENT BINDING OF MITOCHONDRIAL RIBOSOMES BY
NTPs 17
Introduction 17
Materials and Methods 19
Results 25
Discussion 33
3 PHOTOAFFINITY LABELING OF 28S RIBOSOMES WITH
8-AZIDO GTP 37
Introduction 37
Materials and Methods 43
Results 49
Discussion 72
4 PHOTOAFFINITY LABELING OF 28S RIBOSOMES WITH
[a P] GTP 79
Introduction 79
Materials and Methods 80
Results 81
Discussion 107

5 ISOLATION, IDENTIFICATION, AND PARTIAL AMINO ACID
SEQUENCE OF S5 AND OTHER RIBOSOMAL PROTEINS 112
Introduction 112
Materials and Methods 115
Results 128
Discussion 159
6 CONCLUSIONS AND FUTURE DIRECTIONS 167
REFERENCES 167
BIOGRAPHICAL SKETCH 179

LIST OF TABLES
Table page
1-1 Protein and rRNA Content of Ribosomes 14
2-1 Noncovalent Binding of Various [a32P]
Nucleotides to 28S Ribosomes 27
2-2 Competition for [a32P]GTP Binding to 28S
Ribosomes by Various Nucleotides 29
2-3 Noncovalent Binding of GTP and 8-Azido GTP to
28S Ribosomes 33
3-1 The Effect of Substituting KCL for NH4C12 and
the Lowering of the MgCl2 Concentration in the
Reaction Buffer on the Noncovalent Binding of
[a P]GTP to 28S Ribosomes 56
5-1 Summary of Peak Identification for RP-HPLC of
28S Ribosomal Proteins 144
5-2 Summary of Ribosomal Proteins and Peptides
Submitted for Gas Phase Amino Acid Sequence
Analysis 155
5-3 Summary of Amino Acid Sequence for Several
Bovine Mitochondrial Ribosomal Proteins and
CNBR Peptides of S5 156
5-4 Comparison of Amino Acid Sequence Found in S5
Corresponding to the E Site Found in Other GTP-
Binding Proteins Involved in Protein Synthesis. 162
v

LIST OF FIGURES
Figure page
1-1 Comparison of amino acid sequences from con¬
served binding regions, A,C,E, and G in GTP-
binding proteins 8
2-1 Noncovalent binding of [a32P]GTP, GMP, ATP,
UTP, and CTP to 28s bovine mitochondrial
ribosomes 26
2-2 Binding inhibition of [ mitochondrial ribosomes by various
nucleotides 28
2-3 Noncovalent binding of [a32P] 8-Azi do GTP and
[8- H]GTP to 28S bovine mitochondrial ribosomes 31
2-4 Binding inhibition of [a32P] 8-Azi do GTP to
bovine mitochondrial ribosomes by GTP 32
3-1 Generation of an aryl nitrene following uv
irradiation of 8-Azido GTP 40
3-2 Photoaffinity labeling of 28S ribosomes with
[a P] 8-Azi do GTP 51
3-3 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes witn [a P]8-Azido GTP.... 53
3-4 Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S
ribosomes with [a P] 8-Azi do GTP 58
3-5 Continued optimizatign gf the intensity and
duration of uv irradiation for photoaffinity
labeling of 28S ribosomes with [yP]8-Azido GTP 60
3-6 The effect of decreasing concentrations of MgCl2
on ohotoaffinity labeling of 28S ribosomes with
[y32P] 8-Azi do GTP 63
3-7 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with increasing
concentrations of [y P]8-Azido GTP 66
4-1 Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S
ribosomes with [a P]GTP 82
vi

4-2
Specificity of the photoinduced radiolabeling of
28S ribosomal proteins by [a P]GTP in the
presence of other nucleotides 85
4-3 Determination of the binding affinity of [a32P]
GTP for the specifically labeled 38 xDa ribosomal
protein by photoaffinity labeling of 28S
ribosomes 90
4-4 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with [a32P]GTP 94
4-5 Determination of the requirement for uv
irradiation to radiolabel 28S ribosomes with
[a 2P] ATP and [« P]GTP 100
4-6 Time course far the uv independent radiolabeling
of S4 with [aftP] and [y P]ATP 104
5-1 Reverse phase high pressure liquid chromatogram
of 1.6 nmol of acetic acid extracted 28S
ribosomes resolved on a wide pore (300 A) silica
bonded butyl column 130
5-2 Two-dimensional PAGE analysis of 28S ribosomal
proteins eluted from a RP-HPLC butyl column
coelectrophoresed with a minimal amount of acetic
acid extracted 28S ribosomes 134
5-3 Immunoblot of 28S bovine ribosomal proteins with
an anti S5 monospecific rabbit sera 146
5-4 Immunoblot of 28S bovine ribosomal proteins with
an anti S5 monospecific rabbit sera 147
5-5 Immunoblot analysis of 28S bovine ribosomal
proteins separated by RP-HPLC with an anti S5
monospecific rabbit sera 150
5-6 Immunoblot analysis of S5 protein isolated by
RP-HPLC 152
5-7 Electrophoretic separation of cyanogen bromide
peptides of S5 154
5-8 Elucidation of a putative oxidized tryptophan
residue found in the 5 PTH amino acid residue
of the 13 kDa CNBR peptide of S5 158
vi 1

5-9 Comparison of putative conserved regions found in
the amino acid sequence of CNBR peptides of S5 to
homologous regions found in other GTP-binding
proteins 161
vi i i

ABBREVIATIONS
Á
.... Angstrom, meter x 10'10
AA
.... amino acid
Ac
.... acetate
ACS
.... American Chemical Society
ADP
.... adenosine 5'-diphosphate
AR
.... autoradiogram
ATP
.... adenosine 5’-triphosphate
ATZ
.... ani1inothiazolinone
AU
.... absorbance unit
8N3GTP
.... 8-Azido guanosine 5’-triphosphate
BCIP
.... 5-bromo-4-chloro-3-indolyl phosphate
bp
.... base pairs
BME
.... 6-mercaptoethanol
°C
.... degrees centigrade
%C
.... percent crosslinker (bisacrylamide)
C4
.... butyl
Ci
.... curie, 2.2 x 1012 dpm
cm
.... centimeter
cm2
.... centimeter squared
CNBR
.... cyanogen bromide
cpm
.... counts per minute
CTP
.... cytosine 5’-triphosphate
cGMP
.... 3'-5'-cyclic guanosine monophosphate
cytoribosome.. cytoplasmic ribosome

d day
Da daltons
DEAE diethyl ami noethyl
DEPC diethylpyrocarbonate
dGTP 2'-deoxyguanosine triphosphate
DPTU N,N'-diphenylthiourea
DTT dithiothreitol
E. col i Escheri chi a col i
EDTA ethylenediaminetetraacetic acid
elF eucaryotic initiation factor
EF-Tu bacterial elongation factor-Tu
EF-Ts bacterial elongation factor-Ts
EF-G bacterial elongation factor-G
EF-1 eucaryotic elongation factor-1
f fraction
Fc constant fragment of IgG molecule
g force of gravity
GDP guanosine 5'-diphosphate
gm grams
GMP guanosine 5'-monophosphate
GMPPCP guanosine 5'-[B,Y~methylene]triphosphate
GMPPNP guanosine 5'-[6,Y_imido]triphosphate
GTP guanosine 5'-triphosphate
h hour
3H tritium
HPLC high pressure liquid chromatography
x

IF-1 bacterial initiation factor-1
IF-2 bacterial initiation factor-2
IF-3 bacterial initiation factor-3
IgG immunoglobulin G
Imm immune
ITP inosine 5'-triphosphate
Kd binding dissociation constant
kDa kilodaltons
Ki binding inhibition constant
m mi 11 i
M molar, moles per liter
mA milliamphere
Mr molecular mass
\i micro
ng microgram
nl microliter
nM micromolar
nW microwatt
mg milligram
min minute
mi toribosome.. mitochondrial ribosome
ml mi 11 i 1iter
mM millimolar
mmol mil limóle
mRNA messenger ribonucleic acid
mt mitochondria
xi

MWM molecular weight markers
n number
NADP nicotinamide adenine dinucleotide phosphate
ng nanogram, grams x 10'9
nm nanometer, meter x 10‘9
nmol nanomole, moles x 10’9
ID one-dimensional
NBT nitro blue tetrazolium
NC no competition
NEM n-ethylmaleimide
ND not detected
NTP nucleotide triphosphate
Org organic
P peak
32P 32phosphate
PAGE polyacrylamide gel electrophoresis
pg pi cograms, grams x 10'12
Pi phosphate
PI preimmune
PITC phenyl isothiocyanate
pmol picomole, moles x 10'12
POPOP l,4-Bis(5-phenyloxazol-2-yl)benzene
PPO 2,5-diphenyloxazole
Prep preparation
PTH phenylthiohydantoin
PVDF poly(vinylidene difluoride) membrane
XI 1

Rib ribosome
Res residue
RF release factor
RP-HPLC reverse phase-HPLC
RNA ribonucleic acid
rRNA ribosomal ribonucleic acid
S stained gels (used in figures)
S Svedberg
S5 small subunit ribosomal protein #5 (or 1 to 33)
SD standard deviation
sec seconds
SE standard error
SDS sodium dodecyl sulfate
SW sperm whale
%T percent total monomer
TBS tris buffered saline
TBSA tris buffered saline with sodium azide
TEA triethanolamine
TEMED N,N,N',N'-Tetramethylethylenediamine
TFA trifluoroacetic acid
TMA trimethyl amine
TP total protein
tRNA transfer ribonucleic acid
Tris-HCL tris-hydroxymethylaminomethane-hydrochloride
Triton X-100.. Octylphenoxypolyethoxyethanol
2D two-dimensional
xi i i

UTP uridine 5'-triphosphate
uv ultraviolet
V volt
X any amino acid
xi v

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
CHARACTERIZATION AND IDENTIFICATION OF A NOVEL GUANINE
NUCLEOTIDE BINDING SITE ON THE BOVINE MITOCHONDRIAL RIBOSOME
by
John Claude Anders
December, 1990
Chairman: Dr. T. W. O'Brien
Major Department: Biochemistry and Molecular Biology
Bovine mitochondrial ribosomes possess a single, high
affinity binding site for GTP and GDP on the small subunit.
This is in contrast to procaryotic and eucaryotic cytoplasmic
ribosomes which do not bind GTP directly but do so to soluble
protein factors which cycle on and off the ribosome. We used
a photoaffinity analog of GTP, 8-Azido GTP and well as GTP to
identify ribosome components associated with the GTP binding
site. 8-Azido GTP binding to the small subunit was saturable,
with an apparent dissociation constant (Kd) of 1.92 jiM,
approximately 126 times higher than that for GTP. Photolysis
of both the photoreactive analog and GTP resulted in the
specific labeling of a single ribosomal protein, S5.
Photolabeling of S5 by both [a32P]GTP, and its 8-azido analog,
was blocked competitively by the addition of excess GTP,
xv

demonstrating the labeling was site specific. Partial amino
acid sequence and analysis of this protein revealed two areas
of amino acid sequence having significant homology to
conserved sequences involved in GTP binding in other GTP-
binding proteins. This protein with a guanine nucleotide
binding site may participate in initiation complex formation
on mammalian mitochondrial ribosomes. These mitochondrial
ribosomes apparently employ a different mechanism for
initiation of protein synthesis than bacterial or eucaryotic
cytoplasmic ribosomes which are unable to interact directly
with GTP.

CHAPTER 1
INTRODUCTION
An Overview of GTP-Bindina Proteins
G-proteins are a central component of the cellular
machinery driving olfaction, vision, control of cell prolif¬
eration, cellular regulation, and protein synthesis (for
reviews see Stryer, 1986; Gilman, 1987; and Allende, 1988).
Their basic function in the first four instances is to mediate
signal transduction between the cell surface and the cyto¬
plasm. This process begins when a ligand binds to the extra¬
cellular portion of a receptor protein. Upon ligand binding,
a conformational change in the receptor allows binding of a
GDP binding protein to the cytoplasmic portion of the same
receptor. This results in the exchange of GDP for GTP on the
membrane bound G-protein. This exchange of guanine nucleo¬
tides is often mediated by protein exchange factors which are
an important site for regulation of these processes. This
exchange results in conformational changes in the G-protein
which results in dissociation of two subunits of the G-protein
heterodimer, commonly called G„Y, leaving an activated Ga-GTP
protein. Activated Ga-GTP now binds another membrane bound
acceptor protein such as cGMP phosphodiesterase, adenylate
1

2
cyclase, or phospholipase C, which results in catalytic
production of a second messenger molecule cGMP, cAMP, or
inositol triphosphate and diacyl glycerol f respectively. These
soluble, second messenger molecules complete the signalling
event by binding to tissue specific intracellular receptors.
In the case of second messengers cGMP and cAMP, they modulate
conductance of various ions through the plasma membrane.
Inositol triphosphate and diacyl glycerol bind to the
endoplasmic reticulum, resulting in release of intracellular
calcium, and to protein kinase C (PKC), forming an active PKC,
respectively.
Another feature of GTP-binding proteins is an intrinsic
GTPase activity which results in hydrolysis of GTP to GDP.
Following activation of the acceptor molecule, the GTP-binding
protein turns itself off by hydrolyzing the bound GTP to GDP
resulting in an altered conformation for the protein. This
results in a reduced affinity and dissociation from the
acceptor protein thereby inactivating the signaling event.
In protein synthesis, GTP-binding proteins function in
a different manner. There is no signaling across a cell
membrane upon ligand binding and no membrane bound G-protein
is involved, as described above. However, the G-proteins in
this system (initiation and elongation factors) do bind GTP.
Binding of GTP results in a conformational change in the
factor that allows it to bind to the ribosome and produce a
competent initiation or elongation complex leading to protein

3
synthesis. Hydrolysis of GTP, bound to these factors,
provides the energy to drive both initiation and elongation in
protein synthesis.
Protein Synthesis
G-Proteins are a key component in protein synthesis in
both procaryotic and eucaryotic systems. The components of
protein synthesis are analogous in both systems, differing
primarily in the increased complexity and numbers of accessary
proteins in the eucaryotic system. These accessary proteins
are involved in regulation of translation in the eucaryotic
system (for review see Moldave, 1985). Many features of
procaryotic ribosomes resemble that of eucaryotic ribosomes
therefore description of protein synthesis, will be largely
based upon the accumulated knowledge of translation in
procaryotic systems.
Translation of information encoded in messenger RNA
(mRNA) begins with the formation of a competent ribosomal
initiation complex. In E. coli. a GTP-binding initiation
factor, IF-2, binds GTP and initiator fmet-tRNA on the small
subunit of the ribosome forming a GTP-ternary complex. The
order of binding is random and IF-2 does not necessarily bind
initiator methionyl t-RNA prior to binding the ribosome
(Gualaerzi, 1977, and 1986). In conjunction with two other
protein initiation factors, IF-1 and IF-3, mRNA binds to
complete the initiation complex. Upon formation of the
complete initiation complex on the small subunit, the

4
intrinsic GTPase activity of IF-2 is activated and bound GTP
is hydrolyzed resulting in the release of GDP-bound IF-2, IF-
1, and IF-3 from the small subunit. The large subunit now
binds to the small subunit with the initiator tRNA held in
proper orientation with respect to the initiator codon
(usually AUG) of the mRNA to form a monosome to start
polypeptide elongation. IF-1 and IF-3 (which are not GTP-
binding proteins) are known to increase the binding affinity
of the GTP-ternary complex (Wintermeyer and Gualerzi, 1983)
and IF-3 also inhibits formation of monosomes (Noll and Noll,
1972).
Eucaryotic initiation factor, eIF-2, functions in a
manner nearly identical to IF-2. The major functional
difference is how GDP on the inactive initiation factor
exchange for GTP. The exchange of GDP for GTP occurs
spontaneously in procaryotes due to relatively low binding
affinities for both nucleotides. In eucaryotes the binding
affinity for GDP is typically several fold greater than for
GTP. An example of this is seen in Xenopus 1 eavis oocytes
(Carvallo and Al 1 ende, 1987) where GDP binds 50 times better
than GTP (Kd= 70 and 3800 nM for GDP and GTP, respectively).
The exchange of tightly bound GDP for GTP, on EIF-2, requires
a guanine exchange factor (GEF, Panniers and Henshaw, 1983)
which regulates initiation of protein synthesis in eucaryotes
(Ochoa, 1983).

5
Three elongation factors are required for protein
elongation, EF-Tu/Ts, EF-G in procaryotes and EF-1/1B, EF-2 in
eucaryotes. Two of these are GTP-binding proteins, EF-Tu, and
EF-G (EF-1, and EF-2 in eucaryotes) while EF-Ts (EF-1B in
eucaryotes) is an exchange factor. Both procaryotic and
eucaryotic elongation factors function in the same manner.
When GTP is bound to EF-Tu (or EF-1) a conformational change
occurs allowing binding of ami noacyl-tRNA to the factor (Crane
and Miller, 1974). Upon binding to the monosome and
positioning of the ami noacyl-tRNA in the P-site, the intrinsic
GTPase activity induces hydrolysis of GTP, and the GDP-bound
EF-Tu leaves the ribosome. The exchange factor, EF-Ts,
catalyses the exchange of GDP for GTP to reactivate the EF-Tu.
Elongation factor EF-Tu, bound with GTP can now bind the
ribosome. This induces the hydrolysis of GTP which provides
the energy to transfer of the ami noacyl-tRNA to the A site.
Once in the A site and the codon-anticodon pairing is correct,
the peptide chain is transferred from the P site (peptidyl
transferase reaction) to the amino group of the newly arrived
amino-acyl-tRNA. This generates a peptidyl-tRNA. Another
GTP-binding protein, EF-G catalyzes the translocation of the
peptidyl-tRNA from A to the P-site. The energy to drive
translocation is provided by the hydrolysis of GTP bound to
EF-G.
Termination of protein synthesis in procaryotes is not as
well worked out as are the initiation or elongation phases.

6
There are three release factors, RF-1, RF-2, and RF-3. RF-1
and RF-2 bind termination codons, UAA or UAG, and UGA,
respectively in mRNA (Tate and Caskey, 1974). The binding of
RF-1 or RF-2 to the ribosome stimulates the binding of the
third release factor, RF-3. This factor binds GTP and it
promotes the dissociation of release factors from the
ribosome, completing the synthesis of the nascent polypeptide.
A eucaryotic RF has been described from reticulocyte
preparations (Konecki et al., 1977) which also binds GTP and
has a GTPase activity.
The common elements seen in all the GTP-binding
initiation, elongation, and termination factors are that they
incur conformational changes upon binding GTP, that allow them
to mediate the binding of ami noacylated tRNAs or other protein
factors to the ribosome. Once completed they hydrolyze GTP to
GDP to provide the energy to release the factor or drive
protein synthesis.
Conserved Amino Acid Sequences Amona GTP-Bindina Proteins
Proteins that bind GTP and GDP comprise a diverse group.
The group includes the polypeptide initiation and elongation
factors, tubulin, transducin, adenylate cyclase receptor
proteins, and a family of ras proteins. The ras genes encode
a family of closely related proteins of approximately 21 kDa
and were initially identified in human lung and bladder
sarcomas (Scolnic et al., 1979; Shih et al., 1980; Papageorge
et al., 1982).

7
Halliday (1984) described four regions of homology among
various G-proteins whose amino acid sequences were known at
the time. The proteins were bacterial elongation factors (EF-
G and EF-Tu of E. coli). human c-H-rasl gene product (c-H-
ras). and yeast c-rassc gene product (figure 1-1). Van Meurs
et al. (1987) expanded this comparison to include the a-
subunits of both Ta rod and Ta cone transducin and the a-
subunits of GTP-binding proteins that modulate adenylate
cyclase, Gi and Gs. All sequence information for these
proteins were obtained from bovine sources. Recently,
several other notable G-proteins have been sequenced, a
bacterial initiation factor (IF2) and even a yeast
mitochondrial elongation factor (EF-Tumt, see figure 1-1 for
references). The same four regions of conserved sequence were
found in all these GTP-binding proteins from bacterial, yeast,
and mammalian sources and are shown in figure 1-1.
Two additional conserved sites for the mono-ADP
ribosylation by bacterial toxins were found in the a-subunits
of transducin and GTP-binding proteins that modulate adenylate
cyclase (for review see Ueda and Hayaishi, 1985). ADP-
ribosylation is a post-translational modification that
utilizes NAD as the donor of the ADP-ribose group and leads to
a decreased rate of GTP hydrolysis. This impaired intrinsic
GTPase activity leaves the GTP-binding protein in a
permanently activated (GTP-bound) state (Cassel and Selinger,
1977).

A
C
E
G
Gi
36
Go
36
Gs
43
T. rod
32
Ta cone
36
c-H-ras
6
Ras^
13
EF-fu
15
EF-G
14
IF-2
394
EF-Tuâ„¢
51
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LLLLGAGESGKSTIV
LVWGAGÍGV|GKSAm
IVWGGG GV GKSAIT
vjGÍjlGSvcgGKTTI
IGIS
VT
AHIDAGKT
MGHVCH
GTIGHViaHGKTT
198
199
221
198
194
55
62
78
85
442
115
MFDVGGQPSE
LFDVGGQFjSE
MFDVGGQRÃœE
MFDVGGQFEE
MjFpVGGQgSE
ildSag
ILDIAG
IVDCPGÍHApY
[iIDIPGJHViDF
FlLEIP
HVDtdP1
216
217
239
212
216
76
83
98
106
463
136
EGVTjAflTTOV
EDVTATIFCV
NDVTAIIFVJV
EGVT CUFIA
EGVgCIIFpA
EG^LOVpAIN
EGFIpWSVT
DGAIJL
DGA
VjVLW AAD
dgaiilwIaIat
264
265
287
260
264
112
118
130
137
494
167
IILFLNKKD
IILFLNKKD
VILFLNK@D
IVLFLNKKD
IVLFLNKKD
vlvIg^nkBd
VWG-NKLD
IIVFLNKCD
KUD
KID
iwfvnkMd
oo
Figure 1-1 Comparison of amino acid sequences from conserved
binding regions, A,C,E, and G (Hal 1 id ay r 1984, and Van Meurs
et al., 1987) for GTP-binding proteins that modulate adenylate
cyclase Gia (inhibitory, Nukada, et al., 1986), Goa (brain; Van
Meurs et al., 1987), Gsa (stimulatory, Robisnaw et al., 1986,
and Michel et al., 1§86), T rod (Tanabe et al.. 1985,
Medynski et al., 1985, and Yatsunami and Khorana. 1985), Ta
cone (Lochrie et al., 1985, and Lerea et al., 198b), c-H-ras
(Capon et al., 1983), ras,c (i. cerevisiae. DeFeo-Jones et aTT7
1983), EF-Tu (E. coli. Jones et al., 1980, and Laursen et al.,
1981), EF-G (E. col i, Ovchinnikov et al., 1982), IF-2 (E.
col i. Saucerdot et al., 1984), and EF-Tumt (i. cerevisiae.
Nagata, et al., 1983). Boxes surround four or more residues
that are identical or conserved. Residues were grouped
according to Dayhoff (1978) conservative categories, C;
S,T,P,A,G; N,D,E,Q; H,R,K; M,I,L,V; F,Y,W.

9
Cholera toxin is a ADP-ribosyltransferase that modifies
the a-subunits of Gs (Cassel and Pfeuffer, 1978) and
transducin (Abood et al., 1982). The ADP-ribosylation site is
arginine specific, located at Arg174 in o-Transducin within the
conserved peptide, Ser-Arg-Val-Lys.
The second enterotoxin, pertussis toxin, modifies the a-
subunits of Gi(Bokoch et al., 1983 and 1984), Go (Hurley et
al., 1984), and also transducin (West et al.,1985). This
second ADP-ribosylation site is located within three residues
of the COOH-terminus of a-transducin at Cys347 (S-glycoside,
Hurley et al., 1984). This site resides within a conserved
peptide, Asp-Cys-Gly-Leu.
Various means have been employed to identify the
nucleotide binding sites of most of these proteins. The first
to be well resolved was elongation factor Tu (EF-Tu) from
Escherichi a coli. by means of X-ray crystallographic studies
(la Cour et al., 1985; Jurnak, 1985). De Vos et al. (1988)
determined the crystal of cellular ras p21 to the same
resolution as EF-Tu, 2.7 A. Essential features of both
structures are similar, only the structure of EF-Tu will be
discussed below.
Three domains of E.col i EF-Tu were found and the best
resolved was the GTP binding domain located at the NH2
terminal portion of the protein, comprising the first 200
residues. The GTP/GDP ligand site was found to be located at
the C00H terminal end of a sheet of four parallel R strands.

10
The guanine nucleotide was in the anti-conformation in respect
to the sugar and found in an unusual location, at the outer
edge of the domain, rather than within a hydrophobic pocket.
The guanine ring is partially buried in a cavity and appears
to be fixed in position by the side chains of four key
residues, Asn135-Lys-Cys-Asp. These residues represent a
highly conserved consensus sequence, of Asn-Lys-X-Asp, found
in region G (figure 1-1) of all GTP-binding proteins. The
ribose was exposed to the solvent. Asn135 and Asp138 lie near
the guanine and can bind the keto and amino substituents of
the guanine ring, respectively. Residues in both the E and G
region reside in two B-strands that interact with one side of
the guanine base (Jurnak, 1985, and la Cour et al., 1985).
Mutagenesis of Lys136, in conserved region G of bacterial
EF-Tu to an Arg or a Glu reduces the ability of the protein to
bind guanine nucleotides by about 20 or 100 fold, respectively
(Hwang et al., 1989). In the ras p21 protein, mutagenesis of
the corresponding Asp116 to Lys or Tyr in this same conserved
region abolishes GTP binding activity in this protein (Shih et
al., 1988).
In regions A and C (figure 1-1) are residues that
interact with the pyrophosphate group. Crystallographic
analysis by Jurnak (1985) and la Cour (1985) revealed that Mg2+
is located close to the B-phosphate of GDP, forming a salt
bridge to Asp80 of EF-Tu. This residue was found in the
conserved peptide, Leu-Asp-Thr-Ala-Gly, in region C. In the A

11
region, the sequence Gly-X4-Gly-Lys-Ser/Thr is found in all
GTP-binding proteins examined so far. Lysine24, in bacterial
EF-Tu, is at the NH2-terminus of a «-helix which has been
postulated to form a positive dipole that partially
neutralizes the negative charge of one the phosphates (Hoi et
al., 1978, Jurnak, 1985). Additionally, Ohmi et al. (1988)
affinity labeled the corresponding Lys16 in the ras oncogene
product p21 with guanosine diphospho- and triphospho-
pryridoxals. These observation suggest that this residue in
the conserved sequence is located in the guanine nucleotide
binding site, close to the 6- or y-phosphate group of the
nucíeotide.
Site directed mutagenesis in a family of closely related
genes, ras, which encode a group of homologous 21 Kda
proteins, has yielded additional information about guanine
interactions with these GTP-binding proteins. In region A the
conserved region Gly10-X4-Gly-Lys-Ser/Thr in ras p21 forms a
loop that interacts with the pyrophosphate group as described
above. Shih et al. (1988) mutated individually the Gly
residues at codons 10 and 15, to Val in ras p21 and found the
GTP binding was reduced by 33 and 1000 fold, respectively.
In Harvey and Kirsten sarcoma viruses, oncogenic ras
genes are found that contain a point mutation in this same
conserved region, resulting in the replacement of Gly12 for Val
(Taparowsky et al., 1982, Tabin et al., 1982, and Reddy et
al., 1982). Other transforming point mutations have been

12
identified and are discussed fully in the review by Barbacid
(1987).
Site directed mutagenesis of Gly12 in the cellular ras
proto-oncogene resulting in the substitution of any other
amino acid except proline for this residue conferred
transforming properties on the ras p21 gene product. The
authors postulated that a-helical structure in this region was
required for transformation (Seeburg et al., 1984). Insight
into the molecular events disturbed by this point mutation was
brought forth by McGrath et al. (1984). They found that this
single point mutation, resulting in the substitution of Gly12
by a Val, not only reduced GTP binding activity but also
impaired the intrinsic GTPase activity of ras p21 protein by
at least 10-fold. The transformation of the protoncogene
product, ras p21, was attributed to this lowered rate of
GTPase activity. Kinetic analysis of the hydrolysis of GTP by
p21 ras revealed that the mutant (Asp12) protein had a lowered
rate of hydrolysis and GDP release, in comparison to that of
the proto-oncogene (Val12), consistent with the reduced GTPase
activity found in this mutant (Neal et al., 1988).
Protein Synthesis in Mitochondria
The mechanism of protein synthesis in mammalian
mitochondria is poorly understood. Mammalian mitochondrial
ribosomes have been generally characterized as procaryotic in
nature due to some shared antibiotic sensitivities (Denslow
and O'Brien, 1974) and that both utilize f-Met-TRNA for

13
polypeptide initiation. However, a number of differences
exist. Susceptibility to lincosamies and macrolides is much
lower than in procaryotes suggesting some components of the
peptidyl transferase center are altered (Denslow and O'Brien,
1978). Additionally, mammalian mitochondrial ribosomes are
not sensitive to the antibiotic kirromycin, which stabilizes
the binding of the EF-Tu-GTP-aminoacyl-tRNA complex to the
bacterial ribosome, thereby inhibiting polypeptide elongation
(Schwartzbach and Spremulli, 1989).
In regard to their fine structure and physicochemical
properties, mitochondrial ribosomes differ considerably from
other kinds of ribosomes. The mass of the mitoribosome is
approximately the same as the procaryotic ribosome, but the
buoyant density is much less. This is due to the fact that
mitoribosomes contain only half the amount of rRNA and twice
the protein as bacterial ribosomes (Matthews, et al., 1982).
The number of ribosomal proteins is also much greater in the
mitoribosome (table 1-1). In addition, mi toribosomal proteins
do not appear to have any obvious homologue in the bacterial
or cytoribosome as identified by electrophoretic or immuno¬
logic cross-reactivity (Pietromonaco et al., 1984 and Matthews
et al., 1982). Based upon this evidence and functional
studies described below, O'Brien and Matthews (1976) have
assigned mitochondrial translation systems to four separate
classes (Protista, fungi, plants, and animals).

14
Table 1-1
Protein and rRNA Content of Ribosomes
Source
Ribosome
(S units)
Number of
Proteins
rRNA
(S units)
Bovine
28S"
33
12S
Liver
39Sb
52
16S
Mitochondria
55SC
85
Procaryotic
30S?
21
16S
E. coli
50Sb
32
23S, 5S
70SC
53
Mammalian
40S*
33
18S
Eucaryotic
60Sb
50
28S, 5.8S,5S
Cytoplasmic
80SC
83
¡“Ribosome small subunit
Ribosome large subunit
cMonosome
The mechanism for binding mRNA in mi toribosomes appears
to differ from that of procaryotic and eucaryotic cytoplasmic
ribosomes. In procaryotes a leader sequence 5' to the
initiation codon (AUG) binds to a complementary sequence on
16S rRNA, called the Shine-Dalgarno (1974) sequence (Steitz
and Jakes, 1975). This mRNA binding site is absent in
mitochondrial mRNAs which often begin with the initiation
codon or have very short leader sequences which have no
complement on 12S rRNA (Clayton, 1984). Eucaryotic mRNAs also
lack any complementary binding to rRNA. However, they do have
a modified 5'-terminal modification, the m7G(5’)ppp cap which
functions as a recognition signal (Shatkin, 1976) for a cap

15
binding protein. Once the cap binding protein binds the
modified mRNA it scans the 5'-untranslated mRNA leader for the
proper initiation codon. Though the mechanism for initiation
codon recognition in the mitoribosome is unknown, Denslow et
al. (1989) did discover that the major interaction of the mRNA
on the mammalian mitoribosome, covered a 30-nucleotide
segment. Also, the binding of authentic mitochondrial mRNAs
required homologous initiation factors.
Heterologous translocases and initiation factors from
several sources were tested to determine if they would
function with mammalian mitoribosomes. With few exceptions,
homologous factors were required to support i_n vi tro poly
(U)-dependent phenylalanine polymerization in mammalian
mitoribosomes. The only heterologous factor that has been
shown to support poly (U)-directed systems in animal
mitoribosomes was EF-Tu from a variety of sources (E. coll, B.
subtil is. and a protist Eualena gracilis. Eberly and
Spremulli, 1985). Denslow et al. (1988) also showed that
bacterial IF-3 could bind to small subunits of mitoribosomes
and inhibit formation of the monosome as it does in E. coli .
Bacterial EF-G (Denslow and O'Brien, 1979) and other trans¬
locases from gram-positive and gram-negative procaryotes,
protist, plants (chloroplast) and eucaryotic cytoplasmic
origins did not support ijn vi tro phenylalanine polymerization
(Eberly et al., 1985). This was surprising in view of the
fact that a postribosomal supernatant from E. coli would

16
function in a poly (U)-directed system in Neurospora crassa
mitochondrial ribosomes (Grandi etal., 1971). This suggested
that mammalian mitochondrial ribosomes may differ signifi¬
cantly from other types of mitochondrial ribosomes in their
interactions with soluble initiation and elongation factors.
The first reported isolation of any elongation factors
from mammalian mitochondria was EF-Tu/Ts complex from bovine
liver by Schwartzbach and Spremulli (1989).
Proposal
GTP binding proteins are an essential component in pro¬
tein synthetic machinery in procaryotic and eucaryotic cyto¬
plasmic systems. Though much is known about protein synthesis
in procaryotes and much more is being discovered about the
eucaryotic cytoplasmic system, little is known about protein
synthesis in mitochondrial systems. In a search for a puta¬
tive mitochondrial elongation factor, O'Brien et al. (1990)
discovered a unique GTP binding activity on the small subunit
of the mammalian mitochondrial ribosome. This GTP-binding did
not occur on soluble initiation or elongation factors, as is
the case for other ribosomal systems, but on the surface of
the mitochondrial ribosome, suggesting a novel mechanism for
initiation of protein synthesis. The objective of this
research was to identify the site of GTP binding on the small
subunit and further characterize this binding activity. Once
identified, the GTP-binding ribosomal protein was purified
and partial amino acid sequence was determined in an effort to
find any shared homologies to other GTP-binding proteins.

CHAPTER 2
NONCOVALENT BINDING OF MITOCHONDRIAL RIBOSOMES BY NTPs
Introduction
Guanosine-5'-triphosphate plays a key role in protein
synthesis in both procaryotic and eucaryotic systems. In both
systems GTP binds to soluble protein factors in a cyclic
manner to promote the initiation or elongation phases of
protein synthesis (Moldave, 1985, and Allende, 1988).
Elucidation of the translation process in mammalian
mitochondria has been difficult. The substitution of
heterologous translation factors from a variety of procaryotic
and eucaryotic sources generally fail to support poly (U)-
directed phenylalanine synthesis with mammal ian mitoribosomes.
Only EF-Tu from a variety of sources was capable of
functioning in mammalian mi toribosomal translation (Eberly et
al., 1985) as discussed above.
In an effort to isolate a putative mitochondrial EF-G
from bovine liver O'Brien et al. (1990) serendipitously found
that [8-3H] GTP binds to salt washed small subunits of
mammalian mitochondrial ribosomes. This is surprising due to
the fact that GTP-binding to activity in both procaryotic and
eucaryotic cytoplasmic translation systems occurs only on
17

18
soluble protein factors that cycle on and off the ribosome in
protein synthesis. This binding activity was resistant to
high salt washes (300 to 500 mM KCL) and independent of any
exogenous factors or fusidic which is known to stabilize EFG-
GTP complex on bacterial ribosomes. Factor independent
binding of GTP to procaryotic (E. col i 1 and eucaryotic
cytoplasmic (Bovine) ribosomes has never been reported and was
not observed when tested in this laboratory (Sonnega et al.
personal communication).
Although no function has as yet been associated with this
activity [8-3H]GTP was found to bind with high affinity (Kd=
15.3 nM) and in a stoichiometric manner to salt washed 28S
ribosomes (O'Brien et al., 1990). The large subunit (39S) and
intact monosomes (55S) did not bind GTP. The binding site was
specific for GTP and GDP and no other nucleotide tested could
compete with [8-3H]GTP binding to the small subunit. Binding
affinity for the 28S ribosome was equally high for GDP (Kd=18
nM).
Nonhydrolyzable analogs of GTP, GMPPNP and GMPPCP, also
bound to the small subunit, but with reduced affinity,
suggesting that binding site is sensitive to alterations in
the structure of the triphosphate side chain. Additionally,
[y32P] GTP was not hydrolyzed following binding to small
subunits. These findings suggest that salt washed ribosomes
do not posses an intrinsic GTP hydrolytic activity. This of

19
course did not rule out the possibility that GTPase activity
may require exogenous factors, as yet identified.
The following study will expand on the above information
in an effort to determine the specificity of this GTP-binding
activity. GTP-binding will be determined using standard
nitrocellulose filter binding assays and under various salt
conditions to determine the optimal ionic conditions for
binding.
Materials and Methods
Reagents
Guanosine[8-3H] 5'-Triphosphate was purchased as a
ammonium salt in a 50% ethanol solution at a specific activity
of 12.6 Ci/mmol and <99% purity (determined by manufacturer)
from Amersham Corporation (Arlington Heights, IL). Adenosine,
cytidine, guanosine, and uridine 5'[a32P] Triphosphate were
purchased as triethyl ammonium salts in aqueous solution at a
specific activity of 410 Ci/mmole from Amersham Corporation.
[a32P] Guanosine monophosphate was purchased also as a
tri ethylammonium salt from New England Nuclear (Boston MA) at
a specific activity of approximately 3000 Ci/mmol and of at
least 90% purity. 8-Azidoguanosine 5’[a32P] Triphosphate, was
purchased as a triethylammonium salt at a specific activity of
4.6 Ci/mmole from ICN Radiochemicals (Irvine, CA). All
unlabeled nucleotide-5'-triphosphates, guanosine di- and
monophosphates were Type I reagents, purchased as sodium salts
from Sigma Chemical Company. The NTPs were isolated from

20
Equine muscle free of vanadate ion and all other nucleotides
isolated from Yeast.
Mitochondrial Ribosome Preparation Solutions
Solution Formulation
A 0.34 M sucrose, 1 mM EDTA, 5 mM Tris-HCL, pH 7.5
B 0.26 M sucrose, 40 mM KC1 , 15 mM MgCl2, 0.8 mM
EDTA, 5 mM 2-mercaptoethanol (BME), 50 jiM
spermidine, 50 nM spermine, 14 mM Tris- HC1, pH 7.5
C 100 mM KC1, 20 mM MgCl2, 0.01% Triton X-100, 5 mM
BME, 20 mM triethanolamine (TEA), pH 7.5
D 40 mM KC1, 15 mM MgCl2, 0.05 mM EDTA , 5mM BME 1.6%
Triton X-100, 0.005% diethylpyrocarbonate (DEPC),
10 mM Tris-HCl, pH 7.5
E 300 mM KC1, 40 mM MgCl2, 5 mM BME, 1.6% Triton X-
100, 0.005% DEPC, lOmM, Tris-HCl, pH 7.5
F 300 mM KC1 , 5mM MgCl2, 5 mM BME, 10 mM Tris-HCl, pH
7.5
G 0.34 M sucrose, 25 mM KC1, 5 mM MgCl2, 5 mM BME,
10 mM Tris-HCl, pH 7.5
Binding Assay Solutions
Reaction Buffer A: 100 mM KC1, 5 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer B: 10 mM NH4C1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer C: 100 mM KC1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME

21
fi-Scintillation Solution
Liquid Scintillation cocktail: 0.5% PPO and 0.05% (w/v) P0P0P
in toluene
Equipment
Beckman LS8000 6-liquid scintillation counter
Millipore 30 place vacuum manifold
Napco Vacuum oven, model# 5831
Preparation of Mitochondrial Ribosomes
Bovine mitochondrial ribosomes were prepared from bovine
liver as described previously (Matthews et al., 1982).
Mitochondria prepared from liver homogenates in buffer A were
treated with 100 jag/ml digitonin to remove microsomal
membranes and then stored at -70°C in buffer B until needed.
To isolate the ribosomes, thawed mitochondria were lysed in
1.6% Triton X-100 and clarified by centrifugation at 10,000
rpm for 45 min in a Beckman JA-10 rotor at 4°C. Bovine
mitoribosomes were concentrated by absorption to DEAE
cellulose in buffer D and eluted with high salt (buffer E).
The crude mitoribosomes eluted from the DEAE cellulose were
layered on a 34% sucrose cushion (20ml) prepared in buffer C
and centrifuged at 35,000 rpm for 24 h in a Beckman type 35
rotor. The pellets were resuspended in buffer C, treated with
1 mM puromycin to remove nascent polypeptides, and centrifuged
in 10-30% sucrose density gradients prepared in buffer F to
yield 28S and 39S subribosomal particles. Dissociation of
subribosomal particles in high salt buffer (buffer F with 300

22
mM KC1) removed any soluble initiation or elongation protein
factors. Subribosomal particles were recovered from gradient
fractions by centrifugation at 100,000 x g in a Beckman Ti 60
rotor for 15 h, resuspended in buffer G and stored at -70°C
until needed.
Binding of GTP to 28$ Ribosomes and Analysis bv Millipore
Filter Assay
Binding activity was determined by means of a Millipore
filter assay. In this assay, ribosomal subunits with bound
radiolabeled NTP were absorbed to nitrocellulose and washed to
remove unbound NTP. The reaction mixture consisted of a fixed
concentration of ribosomal protein (0.1 pM) with varying
concentrations ( 0.05 to 5 nM) of radiolabeled NTP, in
reaction buffer (A, B, or C). The specific activity of the
radiolabeled NTP (specific activity was typically 410 Ci/mmol)
was reduced by dilution with the appropriate unlabeled NTP
just prior to use. The final volume was adjusted to 50 jil
with reaction buffer in a 1.5 ml conical plastic eppendorf
tube and incubated for 5 min on ice. The reaction was stopped
by the addition of 0.5 ml of ice cold reaction buffer and
immediately transferred to a wetted nitrocellulose filter.
The filter was in a Millipore 30 place manifold under vacuum
to remove the aqueous buffer. Another 0.5 ml volume of
reaction buffer was used to rinse out the reaction tube and
also passed through the nitrocellulose filter. The filters
with bound protein were immediately washed with an additional

23
8 ml of ice cold reaction buffer also under vacuum. The total
time for the wash steps was approximately 15 seconds.
The nitrocellulose filters were placed in empty 20 ml
scintillation vials and dried under vacuum and at 70°C for
approximately 1 h. The dried filters were solubilized in 5 ml
of scintillation cocktail solution (0.5% PPO, and 0.05% w/v
P0P0P in toluene). The filters were counted in a b-
scinti11ation spectrometer (Beckman, model #LS8000) at a
counting efficiency of 21% and 88% for 3H and 32P,
respectively. To determine the extent of nonspecific binding
of radiolabeled NTPs to nitrocellulose, control incubations
were done in an identical manner, but in the absence of
ribosomes. The amount of radiolabeled NTP that nonspecific-
ally bound to the filter was then subtracted from the corres¬
ponding treatment. This nonspecific binding was typically <
1% of the experimental treatments. Additionally, total
amounts of radiolabeled NTPs (the same concentration and
specific activity of radiolabeled NTP used in the experiment)
were determined for each experiment. This was done by pipet¬
ting an appropriate amount of radiolabeled NTP onto a wetted
nitrocellulose filter without washing, drying, then counting.
Binding data were plotted the aid of personal computer
using the Enzfitter Kinetics program by Leatherbarrow (1987).
Data was expressed as pmol of radiolabeled NTP bound per pmol
of ribosomes (R) versus the concentration of free NTP (C)

24
using an equation for ligand binding from Edsall and Wyman
(1958) as shown below:
R = n (C) / Kd + C
where: C= concentration of free ligand; Kd= dissociation
constant of ligand; and R= pmol ligand bound/ pmol ribosome
The binding data was linearly transformed by Scatchard
analysis (Scatchard, 1949) by the following equation:
R/C = -1/Kd (R) + n/Kd
where: R= pmol ligand bound / pmol ribosome; Kd= binding
dissociation constant* n= number of binding sites; C=
concentration of free ligand
Binding inhibition studies were done by incubating 0.1 jiM
28S ribosomes with increasing concentrations of (0.5 to 10 nM)
unlabeled NTP for 1 min in reaction buffer for 1 min on ice
under dim light. [a32P]GTP is then added at a saturating
concentration (0.1 *iM) for 5 min under the same conditions.
The samples are then washed and counted as described above for
the amount of [a32P]GTP that is bound to the ribosome in the
presence of increasing concentrations of NTP.
Binding inhibition data were plotted as percent of
control (Y) versus inhibitor concentration with the aid of
personal computer using the Enzfitter Kinetics program by
Leatherbarrow (1987). A modified equation for binding inhibi¬
tion (Cantor and Schimmel, 1980) was used and is shown below:
Y= l-[ l-r(I/Ki)/ 1+r[1+I/Ki] ]xl00
where: r= Kd/A=constant (Kd for GTP= 15.2 nM and A=
concentration of GTP); Y=l-vi/vo (vi/vo = percent inhibition).

25
Results
Binding of NTPs to 28S Ribosomes
O'Brien et al. (1990) found that [83H]GTP binds with high
affinity to 28S ribosomes and appears to be specific for GTP
and GDP only. To further investigate the nucleotide
specificity of binding for the small subunit, a number of NTPs
were tested for their ability to bind to ribosomes and also
their ability to compete for [a32P]GTP binding to the 28S
ribosome. Millipore filter binding assays (figure 2-1 and
table 2-1) confirmed that [a32P]GTP did bind to 28S ribosomes
with high affinity (Kd of 14.3 nM) and in nearly unit
stoichiometry which agrees with the result (Kd = 15.3 nM) of
O'Brien et al. (1990). The only other [a32P]NTP tested that
was found to bind to this ribosome in a stoichiometric manner
was GMP. GMP binds with approximately 800 times lower
affinity to the 28S ribosome than GTP. [a32P]ATP, UTP, and CTP
did not bind to the 28S ribosome to any appreciable degree.
Binding inhibition studies were done to determine which
nucleotides could displace [a32P]GTP from the 28S ribosome.
Denslow et al. (personal communication) found that of the NTPs
(GDP, ATP, CTP, and UTP) tested only GDP was able to compete
for [a32P] GTP binding. In this experiment additional
nucleotides were tested to determine the structural
requirements for this GTP binding activity. In agreement with
the results of O'Brien et al. (1990), GDP did compete strongly
for [a32p]GTP binding to the ribosome with a resulting Ki of

26
ui
2
o
CO
o
m
cu
Q
z
3
o
m
if •
1
• GTP
♦ GMP
â–² ATP
A UTP
â–¡ CTP
0*4-^ "*â– 
-—Psl
—B
i i i i i 1 1 1 1 1 1
0.0 2.0 4.0 6.0 8.0 10.0
FREE NTP (X 106M)
Figure 2-1 Noncovalent binding of (a P]GTP, GMP, ATP, UTP,
and CTP to 28S bovine mitochondrial ribosomes. Binding of the
radiolabeled nucleotides to the ribosomes was determined by
the Mi 11ipore filter binding assay. The same ribosomal
preparation was used for all nucleotides and the assay was
done in reaction buffer A (100 mM KCL, 5 mM MgCk, 10 mM Tris-
HC1, pH 7.4, and 1 mM BME) as described in Methods. The
initial specific activity of the [a P]NTPs was 410 Ci/mmol and
was diluted approximately 160 fold with unlabeled NTP to a
specific activity of 2.15, 1.67, 3.39,and 3.07 Ci/mmol for
GTP, ATP, CTP, and UTP, respectively just prior to use.
[a P]GMP was initially at a specific activity of 3000 Ci/mmol
and diluted with GMP to a specific activity of 10.2 Ci/mmol
just prior to use. The concentration of the ribosomes was 0.1
nM and the radiolabeled nucleotides varied from 0.05 to 10 uM.
Each data point represents the mean of duplicate samples
except for [a P]GTP where individual data points are shown
without averaging. Refer to table 2-1 for Scatchard analysis
of this data and to Methods for additional experimental
detai 1s.

27
9.2 nM (figure 2-2 and table 2-2) suggesting that GTP and GDP
bind to the same site on the 28S ribosome equivalently.
Additionally, ITP and GMP were able to compete to a lesser
extent, with binding inhibition constants (Ki) of 124 and 1140
nM, respectively.
Table 2-1
Noncovalent Binding of Various fa32Pl Nucleotides to 28$
Ribosomes
Nucíeotide
(Kd ± SE)a
(nM)
Number Binding
(n ± SE)
GTP
14.1 ± 2.3
0.80 ± 0.02
GMP
11,500 ± 1,300
1.00 ± 0.06
ATP
77 ± 44
0.09 ± 0.01
CTP
ND
ND
UTP
1,200 ± 990
0.07 ± 0.02
aScatchard analysis of noncovalent binding of [a P]NTP and
[a P]GMP to 28S ribosomes shown in figure 2-1. Data
represents binding dissociation values (Kd) and standard error
(SE) from a single binding experiment. ND: Not detected
The ionic conditions used in which these binding assays
were modified from those used in earlier work by O'Brien et
al. (1990) in order to determine optimal stringencies for the
study of this GTP binding activity. The original work by
O'Brien et al. (1990) used reaction buffer B (10 mM NH4CL, 20
mM Mg2CL2, 10 mM Tris-HCl, pH 7.4, and 5 mM BME) which was

28
o
cu
t—
z
o
o
ÃœJ
o
ai
Ld
CL
Figure 2-2 Binding inhibition of [a32P]GTP to bovine
mitochondrial ribosomes by various nucleotides. Displacement
of bound [a P]GTP (0.l|iM) from ribosomes (0.1 jiM) by
increasing concentrations (0.5 to 10 jiM) of competing
nucleotide was done in reaction buffer A as described in
Methods. The [a P]GTP was diluted with GTP to a specific
activity of 21.b and 14.8 Ci/mmol just prior to use for two
separate binding inhibition experiments (GDP, GMP,
dGTP,ATP,CTP, and GDP, UTP, ITP respectively). The same
preparation of ribosomes and chemical lot of [a P]GTP was
used in both experiments. The binding of [a P]GTP to
ribosomes in the presence of competing unlabeled nucleotide
was compared to binding in the absence of the competing
unlabeled nucleotide and represented as percent of control.
The data are single data points and each curve represents a
single experiment except for GDP where each data point is a
mean of two experiments conducted on consecutive days. Refer
to table 2-2 for analysis of binding inhibition for each
nucleotide described above.

29
Table 2-2
Binding Inhibition for fcx32Pl GTP Binding to 28S Ribosomes bv
Various Nucleotides
Nucleotide
Ki ± SEa
(nM)
GDP
ITP
GMP
dGTP
CTP
UTP
ATP
9.2 ± 1.0
124 ± 19
1,140 ± 270
NC
NC
NC
NC
aBinding inhibition (Ki) of various nucleotides to compete for
aP]GTP binding to the 28S ribosome. Data represents values
Tom a single binding experiment and standard error, shown in
figure 2-2, for a single binding inhibition experiment.
NC: No binding inhibition
developed by Bodley (Bacca, et al., 1976) for the study of GTP
binding to bacterial EFG. Reaction buffer A (100 mM KC1, 5 mM
MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME) was developed
during the progress of the present study and was used in the
above binding and inhibition binding studies. The development
of these ionic conditions will be described later in this
paper. At this point it suffices to say that GTP binds
specifically to the 28S ribosome and quantitatively in the
same manner over a wide range of ionic conditions and
stringencies.

30
Both r
8-3HlGTP and Ta32Pl 8-Azi do GTP Bind to the Same Site on
the 28
S Ribosome
In order to determine if the 8-Azido analog of GTP would
be a suitable photoaffinity probe for labeling the GTP binding
site on the 28S ribosome, filter binding and binding
inhibition studies were performed. In an experiment directly
comparing binding of [a32P]8-Azido GTP and [8-3H]GTP to 28S
ribosomes, the GTP analog did bind to the small subunit with
an apparent dissociation constant of 1.35 jiM (figure 2-3).
The dissociation constant for [8-3H]GTP was 18.1 nM which is
in close agreement with that found by O'Brien et al. (Kd=15.3
nM, 1990) and both bound in unit stoichiometry.
A mean dissociation factor for [a32P]8-Azido GTP and
[a32P]GTP have been determined (table 2-3). These values were
1.92 ± 0.84 nM and 15.2 ± 8.64 nM, respectively. Both
compounds bound to the ribosome in a stoichiometric manner
(1.2 ± 0.4 and 0.84 ± 0.17 sites occupied per ribosome,
respectively). These data suggest that the GTP analog binds
to the 28S ribosome with an affinity approximately 125 times
less than GTP. These mean Kd values for 8-Azido GTP and GTP
will be used throughout the remainder of this paper.
GTP was found to compete with [a32P] 8-Azi do GTP for
binding to the 28S ribosome with an apparent Ki of 106 nM
(figure 2-4). This suggests that GTP and its photoreactive
analog bind to the same binding site on the 28S ribosome.
These data imply that [a32P]8-Azido GTP could be used as a
photoaffinity probe for the identification of the GTP binding
site on the 28S ribosome.

31
0.0 0.2 0.4 0.6 0.8
FREE NTP (X 106M)
Figure 2-3 Noncovalent binding of [a 2P]8-Azido GTP and [8-
HJGTP to 28S bovine mitochondrial ribosomes. Binding of the
radiolabeled nucleotides to the ribosomes was determined by
the Mi 11ipore filter binding assay as described in Methods.
The same ribosomal preparation was used for both nucleotides
and the assay was done in reaction buffer B (10 mM NH,C1, 20
mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME) as described
in Methods. The specific activity of [8-H]GTP and [a P]8-
Azido GTP was 3.1 and 0.98 Ci/mmol, respectively. Each data
point represents the mean of duplicate samples. Scatchard
analysis for the binding of GTP and its 8-Azido analog to 28S
ribosomes revealed an apparent Kd of 18.1 and 1.35 *iM,
respectively.

32
o
o
X
o
Od
o
o
z
ÃœJ
o
Od
ÃœJ
Q_
INHIBITOR CONCENTRATION (X 106M)
Figure 2-4 Binding inhibition of [a32P]8-Azido GTP to bovine
mitochondrial ribosomes by GTP. Displacement of saturating
concentration (1.67 jiM) of [a P]8-Azido GTP from 0.083 jiM
ribosomes by increasing concentrations (0 to 16.7 ^M) of GTP
was done in reaction buffer B as described in Methods. The
specific activity of [a P]8-Azido GTP was 0.98 Ci/mmol The
same preparation of ribosomes and lot of [a P]8-Azido GTP was
used in this experiment and the one described in figure 2-3.
The binding of [a P]8-Azido GTP to ribosomes in the presence
of competing unlabeled nucleotide was compared to binding in
the absence of the competing unlabeled nucleotide and
represented as percent of control. The data are mean values
from duplicate samples and each curve represents a single
experiment. GTP was found to compete for [a P]8-Azido GTP
binding to 28S ribosomes with an apparent Ki of 106 nM.

33
Table 2-3
Noncovalent Binding of GTP and 8-Azido GTP to 28S Ribosomes
Nucleotide Reaction (Kd ± SD) Binding Sites
Bufferc (nM) (n ± SD)
[8-3H & a32PlGTPa A 15.2 ± 8.64 0.84 ± 0.17
[a32P]8-Azido GTPb B 1,920 ± 840 1.20 ± 0.40
aData for the binding of [8-3H] and [ are the mean and standard deviation of / experiments.
bData for the binding of [a32P] 8-Azi do GTP to 28S ribosomes are
the mean and standard deviation of 3 experiments.
formulations for reaction buffers A and B are listed in
Materials
Piscussion
There is one (stoichiometric) nucleotide binding site on
the 28S ribosome and it is specific for GTP and GDP.
Additionally, O'Brien et al. (1990) suggested that the binding
may occur on the interface of the ribosomes because [8-3H]GTP
did not bind to 55S monosomes. However it is not ruled out
that monosome formation induces a conformational change in the
GTP binding site, preventing binding of the nucleotide.
Only GTP and GDP bind the small subunit, and they do so
equally and with high affinity. Most GTP binding proteins
bind GDP more tightly than GTP, requiring an exchange factor
in order to regenerate the active GTP-bound protein. However

34
there are several instances where GTP-binding proteins do have
equal affinities for both GTP and GDP, such as the photo¬
receptor G-protein of frogs (Robinson et al., 1986), E. coli
EF-G (Kaziro et al., 1978), and p21 ras protein (Shih et al.,
1988).
The structural requirements for nucleotide binding in the
GTP/GDP binding site on the 28S ribosome were elucidated from
binding and binding inhibition assays as described above. No
difference were seen in binding affinities for GTP vs GDP
suggesting that the loss of the y-Phosphate does not perturb
binding. However the loss of the B-phosphate as seen in GMP
does decrease binding affinity by at least 100-fold (table 2-
2). Additionally, O'Brien et al. (1990) reported that
nonhydrolyzable analogs of GTP, GMPPNP and GMPPCP, do bind the
28S ribosome, but with much reduced affinity. This was
attributed to alteration of the PB-X-P angles in these analogs
perturbing the binding interactions with the pyrophosphate
group (Yount, 1971).
Substitutions on the guanine ring also appear to be
poorly tolerated (see figure 3-1 for ring numbering system).
The substitution of the purine by a pyrimidine ring as seen
for CTP and UTP completely abolishes the ability of the
nucleotide to bind to or compete for [a32P]GTP binding to the
28S ribosome (table 2-1 and 2-2, respectively). This suggest
that functional groups on the base are misaligned in the
single ring base, preventing interactions with the binding

35
site. Inosine triphosphate did compete partially with
[a32P]GTP binding to the small subunit suggesting that the loss
of the amino group at C2 of GTP can be tolerated to some
extent. However the loss of the same amino group and the
substitution of an amino for the keto group at C6 as in ATPf
abolished all binding activity, suggesting that the keto group
was required for binding. Both the keto and amino group of
the guanine base have been shown in crystallographic studies
to bind to Asn and Asp residues, respectively in the highly
conserved G region (figure 1-1) in bacterial EF-Tu and ras p21
(Jurnak, 1985, la Cour et al., 1985, and De Vos, et al.,
1988).
Alterations of the ribose moiety are not tolerated
either. dGTP did not displace [a32P]GTP from the binding site,
indicating the importance for the hydroxyl group at C2, on the
ribose. O'Brien et al. (1990) also reported that periodate
cleaved GTP did not bind the small subunit suggesting that any
distortion in the conformation of the ribose affects its
binding. Additionally, these investigators found that n-
ethylmaleimide (NEM), a sulfhydryl modifier had no effect on
GTP binding activity, suggesting that a cysteine residue is
not located near critical GTP-binding residues.
The addition of the azi do (N3) moiety at C8 of the guanine
base reduces the affinity of this GTP analog for the small
subunit (Anders et al., 1989). This could be attributed to
stearic hindrance of the planar azido group and a change to

36
the syn conformation of the base preferred by 8-azido purines
(Colman, 1983). In crystallographic studies of EF-Tu and ras
p21 the GTP molecule resides in the GTP binding pocket in the
anti conformation (Jurnak, 1985, la Cour et al., 1985, and De
Vos et al., 1988) suggesting this conformational change could
contribute, at least in part, to the lowered affinity of this
GTP analog for the 28S ribosome.
This nucleotide binding activity on the ribosome, which
is highly specific for GTP and GDP, is consistent with other
GTP-binding proteins. GTP-binding proteins bind GTP in the
nM to low *iM concentration range and GDP commonly in the low
nM range (see reviews by Stryer, 1986; Gilman, 1987; and
Allende, 1988). They also have a single nucleotide binding
site that is specific for GTP and GDP. In a comparison of
nucleotide specificity, number of binding sites, and binding
affinity, 28S ribosomes bind GTP and GDP in a similar manner
as reported for other GTP-binding proteins. The only possible
difference is that an intrinsic GTPase activity has not yet
been observed on the salt washed 28S ribosomes. This does not
rule out the possibility that an exogenous factor(s), as yet
unidentified, may be required for GTP hydrolysis.

CHAPTER 3
PHOTOAFFINITY LABELING OF 28S RIBOSOMES WITH 8-AZIDO GTP
Introduction
Photoaffinitv Labeling of GTP-Bindina Proteins
Labeling receptor molecules with photoreactive analogs of
effector molecules has become a popular method in elucidating
the mechanisms by which their natural analogs regulate
biological phenomena. The chemical approach to receptor site
labeling is still used but suffers from the necessity to
contrive a molecule that can bind in sufficient concentration
to ensure preferential reaction with suitable residues that
can react with the modified ligand. Most of these reagents
are electrophiles which leads to selective incorporation that
may not reveal the residues in close proximity to the binding
site of the 1igand.
Photoreactive ligands can be constructed with addition of
one or just a few photosensitive constituents available today.
The photoreactive moieties commonly used are either diazo- or
azido-derivatives of their natural ligands (i.e.,ATP, cAMP,
GTP, cGMP etc.) and which should bind their receptor, assuming
sufficient latitude existed in the ligand-receptor recognition
37

38
process. They remain unreactive until irradiated with light
by the investigator. Irradiation of the diazo- or azi do-
purine analog provides the energy to form a reactive carbene
or nitrene radical, respectively, that may form a covalent
bond to residues at or near the binding site in a nonselective
manner. These reactive intermediates do display some
electrophilic character. However, «-carbonyl carbenes and
aryl ni trenes, which have been used extensively to identify
nucleotide binding proteins, display wide chemical reactivity
by being able to insert into C-H bonds and other heteroatomic
bonds (Cooperman, 1980).
The ability to control the labeling event and provide
relatively nonselective incorporation of modified ligands has
made this a popular technique for the identification of purine
binding proteins. With the development and commercial
production of 8-Azido analogs of ATP, cAMP, GTP, and cGMP by
the methods of Haley, (1976), and Czarnecki et al. (1979), the
use of these reagents has increased. 8-Azido GTP was used in
this study. Therefore, the following discussion will be
limited to the nitrene, and not carbenes, even though the two
are of the same isoelectronic specie (six valence electrons)
and their chemistries are expected to be qualitatively
similar. Some examples are; azido analogs of GTP have been
used as photoaffinity probes to study GTP-subunit interaction
in adenylate cyclase (Pfeuffer, 1977), tubulin (Potter and
Haley, 1983), transducin (Kohnken and Me Connell, 1985), and

39
bacterial EF-G (Girshovich and Kurtskhalia, 1979). The number
of applications of these reagents is vast and the reader
should consult reviews by Bayley and Knowles, 1977; and
Chowdry and Westheimer, 1979).
The photoreactive azi do compounds remain unreactive in
the absence of light, thus allowing the investigator to bind
the ligand to the binding site before activating the
photoreactive reagent. Once bound, the sample is irradiated
with uv light and a molecule of N2 is photoeliminated and a
reactive nitrene forms (figure 3-1). This nitrene is highly
reactive (Turro, 1980; Bayley and Knowles, 1977; and Chowdhry
and Westheimer, 1979) and it can insert into heteroatomic
bonds of residues at or near the binding site, in a
nonselective manner (Richards and Konigsberg, 1977). The
type of bond formed is dependent upon the nature of the
nitrene. The nitrene comes in two forms; a very reactive
singlet nitrene that prefers to insert into 0-H and N-H bonds,
and the less reactive triplet nitrene that prefers C-H bonds
(Bayley and Knowles, 1977). The nitrene atom in both species
is linear in shape and the orbital occupancy is two
fi 11 ed(sp2)4 orbitals for the singlet, and one filled (sp2)2
and two p orbitals (px)1 and (pz)1 for the triplet nitrene
(Turro, 1980). The singlet nitrene can covalently attach
itself in a single step reaction such as the addition to a C=C
bond, nucleophilic attack of N-H or 0-H bonds, or direct
insertion into C-H bonds in the backbone or si dechai ns of

40
AZIDE FORM
NITRENE FORM
Figure 3-1 Generation of an aryl nitrene following uv
irradiation of 8-Azido GTP. Formation of the reactive nitrene
is accompanied by the photo-elimination of a molecule of N2.

41
residues in the polypeptide residing near the nitrene while in
the binding site. The triplet nitrene requires a coupled
reaction before a covalent bond can form. The first reaction
can be an abstraction of an electron from a C-H bond. This
generates a short lived radical ( CH3) that can couple with
reduced nitrene to form a useful covalent bond or form some
other product. The only other major reactions are
rearrangements, to which aryl nitrenes are not susceptible
(Bayley and Knowles, 1977). The 8-Azido purine analogs do not
form tetrazoles (an isomer that forms a third five membered
nitrogen ring attached at C8 to C9 of the purine ring) in
aqueous solvents to any extent (Haley, B., personal communi¬
cation). These isomers of 8-Azido ATP or GTP are poor photo¬
reactive reagents and would quench photoaffinity reactions.
The productive covalent attachment of a ligand to a
receptor by photololysis is a competition between the rates of
photoincorporation and rates of decomposition plus
decomposition of the reagent. The rate of photoincorporation
consists of the formation of the reactive nitrene which in
turn must react with surrounding amino acid residues to insert
into heteratomic linkages such as C=0, C-S, C-N, C-H, N-H or
water. The estimates for this total reaction rate in aryl
nitrenes are relatively long, approximately 10'4sec when
measured in a soft polystyrene matrix (Reiser et al., 1968).
This suggests that if binding affinities are strong enough, at
least in the millimolar range, then the ligand should reside

42
long enough in the binding site for productive photoinduced
covalent binding to the receptor (Chowdry and Westheimer,
1979; and Potter and Haley, 1983. Additionally, the stronger
the binding affinity of the ligand for its site, fewer
residues will be available for interaction with the nitrene
increasing the likelihood of covalently labeling a specific
residue at the binding site.
Other concerns in photoaffinity labeling, besides binding
affinity as discussed above, are the temperature of incubation
and photolysis, ionic strength, pH, protein concentration,
intensity of photolyzing light and its affect upon the
receptor molecule. In the vast majority of photolabeling
experiments described in the literature, the incubation and
photolysis were done near 0°C and under dim red light to
prevent decomposition of the azi do compound. This was done in
most instances to minimize the rates of exchange thereby
increasing the residency of the radiolabeled ligand in the
binding site and to dissipate heat from the irradiation by uv
light. The ionic strength and pH should be optimal to ensure
not only tight binding of ligand to the receptor but also that
photoincorporation is not diminished in some manner. In the
following study I will describe the photoincorporation of [a
and y32]8-Azido GTP in 28S ribosomes. I will show that the GTP
analog binds specifically to a single ribosomal protein and
will describe the development of optimal photolabeling
conditions for this reaction.

43
Materials and Methods
Reagents
Both [a32P]8-Azido GTP and [y32P]8-Azido GTP were obtained
as triethyl ammonium salt at specific activities of 6.7 and
10.2 Ci/mmole, respectively, from ICN radiochemicals (Irvine,
CA). GTP was obtained from Sigma Chemical Co. as a Type I
grade reagent.
Binding Assay Solutions
Reaction Buffer A: 100 mM KC1, 5 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer B: 10 mM NH4C1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Reaction Buffer C: 100 mM KC1, 20 mM MgCl2, 10 mM Tris-HCl,
pH 7.4, and 1 mM BME
Polyacrylamide Gel Solutions
Laemmli Sample Buffer: 2% SDS, 5% 2-BME, 10% glycerol, and
62.5 mM Tris-HCl, pH 6.8
ID-PAGE Tank Buffer: 24 mM Tris-HCl, pH 8.3, 92 mM Glycine,
0.2% SDS and 0.1 M sodium thioglycolate
2D-PAGE Sample Buffer: 9 M urea, , 10 mM 2-BME, 60 mM
potassium acetate, pH 6.7, and 0.01% aminoethanethiol
2D-PAGE Prep. Buffer: 5 M urea, 2% SDS, and 10 mM
sodium phosphate, pH 7.2
2D-PAGE Pre-Run Buffer: 9 M urea, 57 mM potassium hydroxide,
and 340 mM HPLC grade acetic acid, pH 5.0

44
2D-PAGE Tank Buffer: 0.5% SDS, 0.1 M sodium phosphate, pH
7.2, 3 mM 2-BME and 0.1 mM thioglycolate.
rRNA Extraction Buffer: 15 mM sodium acetate, pH 5.0, 100 mM
sodium chloride, 5 mM disodium EDTA, and 2% SDS
rRNA Run Buffer: 0.5 M Tris-HCl, pH 8.3, 0.5 M boric acid,
and 10 mM EDTA.
Photolabelino of 28S Ribosomes with Radiolabeled 8-Azido~GTP
The conditions used for photoincorporation of
radiolabeled 8-Azido-GTP were nearly identical to those used
for the Mi Hi pore filter assay. The final reaction mixture
contained 28S ribosomes (0.5 and 1.0 nM for ID and 2D-PAGE
analyses, respectively) and radiolabeled NTP at varying
concentrations (0.05 to 6 nM) and GTP (5 and 10 nM for ID and
2D-PAGE analyses, respectively) if a binding site blocker was
desired. The order of addition was important. The ribosomes
and GTP (if appropriate) were mixed in the reaction buffer (A,
B, or C) and incubated on ice and in the dark for 5 min. The
radiolabeled NTP dissolved in reaction buffer was then added
to a final volume of 30 or 100 ^1 for ID or 2D-PAGE analyses,
respectively and incubated on ice in the dark for an
additional 15 min.
Photolysis was performed with a hand held ultraviolet
lamp with a purple filter that allowed transmission at 254 or
377 nm. Irradiation with 254 nm light was done in all cases
and at varying distances directly above the samples resulting
in intensities of 400 to 800 nW/cm2 for photolabeling with

45
8-Azido-GTP or GTP, respectively. The intensity of the uv
light was determined with the use of Spectroline digital uv
intensity meter (model # 254X). In initial photolabeling
experiments with [a32P]8-Azido GTP, 2500 ^W/cm2 was used.
Irradiation of samples was allowed to proceed for 5 to 10 min
typically, on ice, in the dark with only dim red light for the
8-Azido GTP and with minimal room light for GTP. Samples were
then prepared for either ID or 2D PAGE analysis.
Polyacrylamide Gel Electrophoresis
Photolabeled ribosomal samples were often resolved on ID
SDS slab gels (1.5 X 160 X 200 mm) as described by Laemmli
(1970). Samples were loaded into sample wells of a
polyacrylamide SDS gel (12% or 15%T, 3.5%C, 0.375 M Tris-HCl,
pH 8.8, and 0.1% SDS; T is the total monomer concentration, C
is the crosslinker, bisacrylamide, in percentage T) with a
stacker gel (3%T, 3.1%C). Electrophoresis was performed in
ID-PAGE tank buffer (24 mM Tris-HCl, pH 8.3, 0.192 M Glycine,
and 0.2% SDS) at an applied constant current of 10 mA for
approximately 14 h at room temperature. Following
electrophoresis the gel was fixed in 50% methanol and allowed
to equilibrate overnight with repeated changes of the
methanol. The gel was then silver stained (Wray et al.,
1981)and refixed in 50% methanol for at least 4 h to ensure
complete shrinkage of the gel before autoradiography was
performed. The stained and fixed gel was then sandwiched
between two layers of wetted Bio-gel wrap and allowed to air

46
dry while clamped in a plexiglás holding frame. The dried gel
was then autoradiographed at room temperature for an
appropriate period of time.
A modified 2D-PAGE system of Leister and Dawid (1975)
was used to determine which ribosomal protein was photolabeled
by either radiolabeled 8-Azido-GTP or GTP. Following
photolabeling as described above, ribosomal samples were
extracted with urea and lithium chloride as described below.
The first dimension was run in tube gels (3 X 110 mm) cast in
siliconized (Sigmacote, Sigma) glass tubing. The acrylamide
concentration of the separation tube gel was 4.6%T, 3.2%C,
with 9 M urea, and 60 mM potassium acetate, pH 4.3. The first
dimension was pre-run in 2D-PAGE pre-run buffer (9 M urea, 60
mM potassium acetate, pH 4.3) to remove the polymerizing
agent, ammonium persulfate (0.1%). A stacker gel (4%T and
3.5%C) was then cast (3 cm long) on top of the separator tube
gel. To each nl of dialyzed sample, 0.25 nl of sample gel
(4%T, and 3.4%C) was added and then cast on top of the stacker
gel. The first dimension was then electrophoresed at a
constant amperage of 0.2 mA/gel toward the cathode in 2D-PAGE
tank buffer (35 mM B-alanine, pH 4.5, and 0.01%
aminoethanethiol) until the tracking dye, pyronine Y, had
entered the separation gel, then the current was boosted to
0.5 mA/gel. The polymerizing agent of both stacker and sample
gel was 0.1% riboflavin. When the tracker dye was within 1 cm
of the end of the tube gel, electrophoresis was stopped. The

47
tube gel was removed from the glass tube, then equilibrated in
2D-PAGE prep, buffer (5 M urea, 2% SDS, and 10 mM sodium
phosphate, pH 7.2) for 30 min while shaking gently at room
temperature.
For the second dimension a SDS slab (1.5 X 160 X 200 mm)
gel (10%T, and 3.5%C in 5.1 M urea, 0.5% SDS, 0.1 M sodium
phosphate, pH 7.2) was used. In preformed wells adjacent to
the first dimension tube gel, molecular weight markers (94,
67, 43, 30, 20.1, and 14.4 kDa) were run. The second
dimension was run at a constant current of 45 mA/gel toward
the anode in tank buffer (0.5%SDS, 0.1 M sodium phosphate, pH
7.2, and 3 mM BME).
The slab gels were fixed in 50% methanol, with several
changes, overnight. The gels were then silver stained (Wray
et al., 1981) and dried in a sandwich of Bio-gel wrap and
prepared for autoradiography.
Electrophoresis of Mitochondrial 12S rRNA
Photolabeling of mitochondrial 12S rRNA, as well as
ribosomal proteins , with [a32P]8-Azido-GTP or [a32P]GTP, was
also examined. Following extraction of the photo!abeled 28S
ribosomes by urea and Li Cl, the rRNA pellet was stored at -
20!C until analyzed. The rRNA pellet was dissolved in an
extraction buffer (15 mM sodium acetate, pH 5.0, 100 mM sodium
chloride, 5 mM disodium EDTA, and 2% SDS) and extracted three
times with an equal volume (100 jil) of equilibrated phenol
followed by centrifugation at high speed in a Beckman tabletop

48
microcentrifuge for 5 min at room temperature. Sodium acetate
was then added to a final concentration of 200 mM to the
pooled aqueous phases. To precipitate the rRNA, 2.5 volumes
of ice cold 95% ethanol was added and the mixture was kept at
-20°C for at least 4 h. The sample was centrifuged for 10 min
in a microcentrifuge at high speed to pellet the rRNA. The
rRNA pellet was washed two more times with 70% ethanol
followed by a final wash with 95% ethanol. The sample was
dried under vacuum for approximately 15 min.
The rRNA pellet was redissolved in a 1:1 ratio of rRNA to
formamide loading buffer (80% formamide, 10 mM EDTA, pH 8, 1
mg/ml bromophenol blue, and 1 mg/ml xylene cyanol FF) prior
to ID urea PAGE. Samples were resolved with molecular markers
of six synthetic poly(A)-tai1ed RNAs ranging in length from
0.24 to 9.49 kb (BRL, Gaithersburg, MD) on a one dimensional
urea PAGE (7.5%T, 3.5%C, 7 M urea, 0.5 M Tris-HCl, pH 8.3, 0.5
M boric acid, 10 mM EDTA, 0.05% TEMED, and 0.017% ammonium
persulfate) with a stacker gel (5%T, 3.5%C). Electrophoresis
was done in rRNA run buffer (0.5 M Tris-HCl, pH 8.3, 0.5 M
boric acid, and 10 mM EDTA) at a constant voltage of 500 V
toward the anode. The gel was then soaked in 0.2 M sodium
acetate, pH 5.0, for 30 min prior to staining with methylene
blue. The stained gel was washed with water, destained in 5%
acetic acid overnight, and then washed with water again just
prior to photography. The gel was dried overnight in a
sandwich of Bio-gel wrap and prepared for autoradiography.

49
Extraction of Ribosomal Proteins for 2D-PAGE Electrophoretic
Analvsis
Ribosomes were extracted in urea-LiCl to remove rRNA from
ribosomal proteins as described by Matthews et al. (1982)
prior to 2D-PAGE analysis. Thawed 28S ribosomes were adjusted
to 9 M urea, 3 M Li Cl with addition of solid urea and Li Cl
with a resulting neutral pH. The samples were stirred
overnight at 4°C followed by centrifugation at 50,000 rpm
(100,000 X g) for 60 min in a Beckman type 65 rotor to
precipitate the rRNA. The supernatant containing ribosomal
proteins was removed and the rRNA pellet was extracted once
again, by stirring for 2 h at 4°C, with the above buffer.
After centrifugation the supernatants were pooled and dialyzed
against 2D-PAGE sample buffer (9 M urea, 0.01%
aminoethanethiol, 10 mM BME, and 60 mM potassium acetate, pH
6.7) overnight at room temperature in preparation for 2D-PAGE
analysis. The rRNA pellets were stored in the type 65
centrifuge tubes at -20°C until needed.
Results
fg32Pl8~Azido GTP Photolabels a 38 kDa Ribosomal Protein in a
Specific Manner.
Filter binding studies suggest that a single and
specific binding site for GTP exists on the mitochondrial
ribosomal small subunit. To identify which ribosomal
protein(s) may be involved a photochemical approach was used.
28S ribosomes were incubated with increasing concentrations
(0.15 to 6.0 nM) of [a32P]8-Azido GTP in reaction buffer B.

50
The samples were irradiated with uv (254 nm) light for 20 min
at 2500 nW/cm2 on ice (and in the dark). The photoaffinity
labeled ribosomes were resolved on a 12% polyacrylamide
Laemmli gel, silver stained and prepared for autoradiography
as described in Methods. Photoaffinity labeling of ribosomal
small subunits with [a32P] 8-Azido GTP yielded a single
specifically radiolabeled 38 kDa ribosomal protein (figure 3-
2A). The addition of excess unlabeled GTP blocked
photolabeling of this protein, suggesting that the binding
site was also specific for GTP (figure 3-2 A). The amount of
radiolabeling increased as the concentration of the [a32P]8-
Azido GTP increased but is approaching saturation at 6^M
[a32P]8-Azido GTP. This is consistent with the apparent Kd of
1.92 jiM (75% of the binding sites on 0.5^M 28S ribosomes
should be occupied at this Kd and concentration of 8-Azido GTP
used in this experiment) determined by filter binding assays
(table 2-3). Several ribosomal proteins with Mr > 40 kDa were
nonspecifically photolabeled at concentrations of [a32P]8-Azido
GTP above the Kd for 8-Azido GTP.
Unambiguous identification of the specifically photo-
labeled 40 kDa ribosomal protein was not possible by one
dimensional gel analysis due to the existence of other 28S
ribosomal proteins of similar molecular weight. Two dimen¬
sional gel analysis of photoaffinity labeled small subunits
was done in order to do so. In figure 3-3 a single ribosomal
protein, S5, was specifically photolabeled with [cc32P]8-Azido

51
8N3GTP(/iM) 0.3
GTP (5/iM) - +
0.15 0.3 0.8 1.5 6.0
-+- + - + - + - +
Figure 3-2 A Photoaffinity labeling of 28S ribosomes with
[a P]8-Azido GTP. Bovine 28S ribosomes (0.5 nM) were
irradiated in the presence of increasing concentrations (0.15
to 6 *iM) of the 8-azido analog of GTP in reaction buffer B.
The samples were irradiated with uv light for 20 min at 2500
iiW/cm on ice under dim red light. The specific activity of
the [a P]8-Azido GTP was 1.35 Ci/mmol just prior to use. In
panel A a representative lane of silver stained (S) proteins
electrophoresed on a 12% SDS-polyacrylamide gel and the
corresponding autoradiogram (AR) are shown. In alternate
assays the GTP binding site was blocked by the addition (+) of
5uM GTP. The Mr markers were as follows: Phosphorylase b (94
kDa), Bovine Serum Albumin (67 kDa), Ovalbumin (43 kDa),
Carbonic Anhydrase (30 kDa), Soybean Trypsin Inhibitor (20.1
kDa), and alpha-Lactalbumin (14.4 kDa). these same Mr markers
were used throughout this entire study. The arrow indicates
the presence of a 38 kDa protein specifically radiolabeled
with [a P]8~Azido GTP. See Methods for additional details.

52
x
o
LlI
X
¡2
CL
Lü
>
LxJ
a:
8N3GTP CONCENTRATION (X 106M)
Figure 3-2 B Densitometric analysis of the autoradiogram for
the specifically radiolabeled 38 kDa ribosomal protein in
figure 3-2 A is shown. Data were the relative peak height
densi tometri c measurements of the 38 kDa band (panel A) in the
presence of increasing concentrations of [a P]8-Azido GTP and
the absence of GTP.

53
Figure 3-3 Two dimensional PAGE analysis of photoaffinity labeled
28b ribosomes with [a P]8-Azido GTP. Bovine Z8S ribosomes (1 *iM)
were irradiated in the presence of 2 *iM [a P]8-Azido GTP (45%
saturation) and in the presence (B) and absence (A) of 10 ^M GTP.
The samples were irradiated with uv light for 10 min at 2500 ^W/cm
on ice as described in Methods. The specific activity of the
[a Pl8-Azido GTP was 1 Ci/mmol just prior to use. Silver stained
2D-PAGE gels are on the left and their corresponding autoradiograms
are on the right. Autoradiograms were exposed for 14 days. The
molecular weight markers used were the same ones described in
figure 3-2. The arrows indicate the presence of the nonspecific-
ally labeled (S4) and the specifically labeled (S5) proteins. See
Methods for additional experimental details.

54
GTP. The presence of excess amounts of unlabeled GTP com¬
pletely blocked photoincorporation of [a32P] 8-Azido GTP into
S5. Another ribosomal protein, S4, was also labeled
nonspecifically, since the presence of excess amounts of GTP
did not block photolabeling of S4.
Development of Optimal Photoaffinitv Labeling Conditions for
8-Azido GTP
The extent of specific photolabeling of S5 by [a32P]8-
Azido GTP was small, especially in comparison to the much
greater nonspecific labeling of S4 (figure 3-3). Optimization
of photoirradiation and ionic conditions was undertaken to
improve the quality of the photoaffinity labeling of 28S
ribosomes by 8-Azido GTP. Optimal conditions were determined
by both filter binding assays and photoaffinity labeling
methods.
The development of optimal ionic conditions focused on
the modification of two components of the reaction buffer B as
developed by Bodley (Bacca, et al., 1976). Bodley developed
this reaction buffer for the study of GTP hydrolysis by EFG in
bacterial systems. One component, NH4C1, is known to be toxic
to peptidyl transferase activity (Denslow and O'Brien,
personal communication) on the large subunit of the
mitochondrial ribosome and its replacement with another
monovalent salt such as KC1 was studied.
In table 3-1 the effect of replacing 10 mM NH4C1 with KC1
on the binding of [8-3H]GTP to 28S ribosomes was examined by

55
filter binding assays. Increasing concentrations of [a32P]GTP
were incubated with 0.1 jiM 28S ribosomes in reaction buffer B
(treatment #1 in table 3-1) as a control, and the test
reactions were in the same reaction buffer but with 10 mM
NH4C1 replaced with increasing concentrations (10 to 100 mM)
of KC1 and decreasing concentrations of MgCl2. The
substitution of NH4C1 by KC1 or the reduction of MgCl2 from 20
to 5 mM did not alter the binding of [a32P]GTP to 28S
ribosomes. The most stringent condition, 100 mM KC1 and 20 mM
MgCl2, supports binding of [8-3H]GTP to the 28S ribosome in an
equivalent manner as controls. This reaction buffer C (100 mM
KC1, 20 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME) and
was used in experiments discussed below.
Potter and Haley (1983) reported that in their
experience with a number of azido-purine analogs, photolysis
at 254 nm and at intensities of 180 to 800 jiW/cm2 for 1 to 10
min caused little if any detectable damage in a variety of
enzymatic systems. In our initial experiments (figure 3-2 and
3-3) these guidelines were exceeded.
The intensity and duration of photoirradiation was
reduced to evaluate the minimal amount of uv irradiation
necessary to adequately photolabel 28S ribosomes with [a32P]8-
Azido GTP. Ribosomes (0.5 nM) were photolabeled in the
presence of 0.75 nM [ approximately 24% saturation of the GTP binding site
(calculation based upon a Kd of 1.92 nM) in reaction buffer B.

56
Table 3-1
The Effect of Substituting KC1 for NfyCL and the Lowering of
the MgCK Concentration in the Reaction Buffer on the
Noncovalent Binding of lot PI GTP to 28$ Ribosomes
eatment
#
NH,C1
fmM)
MgCl,
(mM)
KC1
(mM)
a
ImM)
Kd
± SE
(nM)
n
±
SE
1 (B)
10
20
75
3.8
±
2.2
0.91
±
0.08
2
-
20
10
75
4.0
±
1.0
0.99
±
0.05
3
-
20
20
85
3.1
±
0.8
0.92
±
0.04
4
-
20
40
105
1.7
±
1.0
0.96
±
0.11
5
-
20
60
125
8.3
±
3.4
0.91
±
0.07
6 (C)
-
20
100
165
5.8
±
2.5
0.87
±
0.06
7
-
10
100
135
2.5
±
0.9
0.92
±
0.06
8 (A)
-
5
100
120
2.7
±
0.8
1.00
±
0.06
Scatchard analysis for the binding of [«32PlGTP to 28S
ribosomes under various ionic conditions by tne Mi 11ipore
filter assay. The data are binding affinities (Kd) and number
of binding sites (n) for a single binding curve. The
concentration of the ribosomes was 0.1 nM and the JV P]GTP
0.02 to 1 liM. The specific activity of the [a PJGTP was
diluted to ¿5.4 Ci/mmol with GTP just prior to use. Reaction
buffers A, B, and C (for complete formulations see Methods)
are anotated in parentheses, beside their respective
treatments . In all reaction buffers ImM BME was used. No
differences were observed with the substitution of NH4C1 in
reaction buffer B (treatment #1) for increasing concentrations
of KC1 (treatments 2 to 6) or decreasing concentrations of
MgCl2 (treatments 7 & 8).
an = total ionic strength
In figure 3-4 A the reduction of 254 nm uv irradiation from
2500 to 800 iiW/cm2 for 10 min reduced the photolabeling of the
38 kDa ribosomal protein (no excess GTP present) by only 47%,
as determined by densitometric analysis (figure 3-4 B). The
amount of labeling was still adequate for analysis. However,
irradiation for 2 min reduced photolabeling of the same 38 kDa
protein by 75 to 90% at 2500 and 800 nW/cm2, respectively.

57
This provided minimal detectable labeling at 2500 ^W/crn2 and
virtually no labeling at the lower level of uv irradiation
even after a 2 week exposure of the autoradiogram. The
pattern of photolabeling remained the same as seen in figure
3-2 A. Photolabeling of the 38 kDa protein by [a32P]8-Azido
GTP was blocked in the presence of excess GTP and not in the
case of the 43 kDa ribosomal protein.
In an continuing effort to find the minimal exposure of
ribosomes with uv light for adequate photolabeling [y32P]8-
Azido GTP was used. This isotope was used because [a32P]8-
Azido GTP was no longer available. [y32P]8-Azido GTP was also
available in higher purity (>98%), more than twice the
specific activity (10.2 Ci/mmole),and at lower cost. Optimal
photoirradiation conditions for the photolabeling of 28S
ribosomes with [y32P]8-Azido GTP were determined in reaction
buffer C (100 mM KC1, 20 mM MgCl2, 10 mM Tris-HCl, pH 7.4, and
1 mM BME). Substoichiometric concentrations of [y32P]8-Azido
GTP (0.43 *iM), corresponding to approximately 18% binding,
were incubated with 28S ribosomes (0.5 ^M) and irradiated at
300 and 800 ^W/cm2 for 4 and 8 min on ice in the dark. The
minimal uv (254 nm) irradiation intensity and duration found
for adequate labeling were 300 nW/cm2 for 10 min (figure 3-5
A and B). These conditions were utilized in subsequent
studies involving this analog of GTP.

58
s
AR
94-
67-
43-
s
.
30-
m
20.1-
14.4-
1
Ribosomes
GTP (5/iM)
Irrad. time (min)
-++ + + + + + +
+ - + - + - +
10 2 10 2
Intensity (/-tW/cm^) 2500
800
Figure 3-4 A Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S ribosomes with
[aP]8-Azido GTP. 28S ribosomes (0.5 uM) were incubated with
0.75 liM [a P]8-Azido GTP (24% saturation) in reaction buffer
B. The specific activity of the [a P]8-Azido GTP was 1.0
Ci/mmol. In panel A is a representative lane of silver
stained ribosomal proteins on the left and the corresponding
autoradiogram on the right. The AR was exposed for 14 days
before development. Tne presence (+) and absence (-) of
ribosomes and unlabeled GTP as well as the intensity and
duration of uv (254 nm) irradiation are noted below the
autoradiogram. The arrow indicates the position of the
specifically radiolabeled 38 kDa protein. For more
experimental details see Methods.

RELATIVE PEAK HEIGHT
59
[a32P]8-Az¡do GTP
800/xW / cm2 2500/xW / cm2
Figure 3-4 B Densitometric analysis of the autoradiogram in
figure 3-4 A is shown. Densitometric analysis was done for
the specifically radiolabeled ribosomal protein (38 kDa) and
the nonspecifical ly labeled protein (43 kDa) ribosomal protein
at varying intensities and duration of uv irradiation. Note
that in the presence (+) of 5 jiM GTP the 38 kDa protein was
not radiolabeled. For additional experimental details see
Methods.

60
GTP (5/i.M ) -+-+-+-+
Irrad.time (min) 4 10 4 10
Intensity (^W/cm^) 300 800
Figure 3-5 A Continued optimization of the intensity and
duration of uv irradiation for photoaffinity labeling of 28S
ribosomes with [y P]8-Azido GTP. 28S ribosomes (0.5 jiM) were
incubated with 0.5 nM [y P]8-Azido GTP (18% saturation) in
reaction buffer C. The specific activity of the [y P]8-Azido
GTP was 8.4 Ci/mmol. In panel A is a representative lane of
silver stained (S) ribosomal proteins on the left and the
corresponding autoradiogram (AR) on the right. The AR was
exposed for 3 days before development. The presence (+) and
absence (-) of unlabeled GTP as well as the intensity and
duration of uv (254 nm) irradiation are noted below the
autoradiogram.

61
032P]8-Az¡do GTP
x
o
ÃœJ
X
¡5
CL
Ld
>
5
UJ
cu
300/¿W / cm2
800/¿W / cm2
Figure 3-5 B Densitometric analysis of the autoradiogram in
figure 3-5 A is shown. Densitometric analysis was done for
the specifically labeled ribosomal protein (38 kDa) and the
nonspecifically labeled protein (43 kDa) ribosomal protein at
varying intensities and duration of uv irradiation. The
presence (+) and absence (-) of 5 nM GTP is indicated below
the figure. In the presence of excess amounts of GTP (+) the
38 kDa protein was not radiolabeled. For additional
experimental details see Methods.

62
No significant differences were seen in the photolabeling
pattern of the two isotopes ( compare figures 3-4 A and 3-5 A)
though the intensity of radiolabeling was greater for the
[y32P] isotope due to its higher specific activity. The
[y32P]8-Azido GTP was utilized in the remainder of 8-Azido GTP
photoaffinity experiments described in this paper.
The last component of the reaction buffer to be modified
was the divalent cation, MgCl2. MgCl2 has been shown by
Denslow et al. (1988) to promote adherence of 28 and 39S
subunits to one another due presumably to salt bridging
effects at higher concentrations of the divalent cation. To
reduce the possibility of nonspecific binding due to salt
bridging of the nucleotide, MgCl2 concentration was reduced
from 20 to 5 mM and photolabeling was examined under these
conditions. The same incubation conditions were used as
described in the above experiment and uv photoirradiation was
done at 300 ^W/cm2 for 10 min. The 38 kDa ribosomal protein
was still specifically photolabeled (figure 3-6 A) by the GTP
analog, [y32P]8-Azido GTP, and a 45% increase in this labeling
was observed by densitometric analysis as the concentration of
MgCl2 dropped (figure 3-6 B) to 5 mM. Since the lowest
concentration of MgCl2 provided a modest increase in
photolabeling of the specifically labeled 38 kDa ribosomal
protein it was adopted for subsequent studies. The amount
oflabeling on the nonspecifically labeled 43 kDa ribosomal
protein was unchanged in the absence of excess GTP but did
increase 47% in the presence of 5 mM GTP.

63
s
AR
94 —
67 —
43 —
vi
30 —
-1
20.1 —
2
14.4 —
V
G TP(5/iM)
-+-+- +
MgCL2(mM)
20 10 5
0.5 uM
uv light
activity of the
Figure 3-6 A The effect of decreasing concentrations of MgCl2
on photoaffinity labeling of 28S ribosomes with [y P]8-Azido
GTP. Bovine 28S ribosomes (0.5 uM) were incubated witn
y P]8-Azido GTP (18% saturation) and irradiated with
;or 10 min at 300 nW/cm on ice. The specific ac
[y P]8-Azido GTP was 6.3 Ci/mmol. The concentration of the
MgCl2 component of the reaction buffer was decreased from 20
mM (reaction buffer C) to 5 mM (reaction buffer A). In panel
A a representative silver stained lane of 28S ribosomal
proteins and molecular weight markers are shown with the
corresponding autoradiogram on the right. The concentration
of the MgCl2 component of the reaction buffer and the presence
(+) and absence (-) of 5 nM unlabeled GTP is shown below the
autoradiogram. The autoradiogram was exposed for 3.5 days
before developing. The arrow indicates the position of the 38
kDa specifically radiolabeled protein.

64
t-
x
CD
UJ
X
á
X
UJ
>
5
Lü
O'
Figure 3-6 B Densitometric analysis of the autoradiogram in
figure 3-6 A. The amount of radiolabeling of the 38 and 43
kDa ribosomal proteins was plotted versus the concentration of
MgClp in the reaction buffer. Note that no radiolabeling of
the 38 kDa protein was detected in the presence of 5 GTP.

65
The three previous experiments suggest that the optimal
ionic composition of the reaction buffer for the binding of
GTP and its 8-Azido analog to 28S ribosomes was 100 mM KC1, 5
mM MgCl2, 10 mM Tris-HCl, pH 7.4, and 1 mM BME and will be
referred to as reaction buffer A in this paper. The optimal
uv photolabeling conditions were 300 jiW/cm2 for 10 min for 8-
Azido GTP.
With these new conditions another attempt was made to
analyze photolabeled ribosomal proteins by two dimensional
PAGE. In figure 3-7 A to F, 28S ribosomes (1 ^M) were
photoaffinity labeled with increasing concentrations of
[y32P]8-Azido GTP (0.3, 1, and 3 ^M) in the presence and
absence of excess (10 |iM) GTP. These amounts of [y32P]8-Azido
GTP correspond to 9, 20, and 60% saturation of the GTP binding
site on the ribosome (based on a Kd of 1.92 tiM). Small
subunit ribosomes were resolved by 2D-PAGE and radiolabeled
proteins were detected by autoradiography.
At all concentrations tested, S5 was specifically
radiolabeled by [y32P]8-Azido GTP (figure 3-7 A to F) and the
radiolabeling increased directly proportional to the
concentration of [y32P]8-Azido GTP (figure 3-7 G). The
presence of GTP completely blocked photoincorporation of the
GTP analog into S5. S4 was also radiolabeled, but in a
nonspecific manner, since excess amounts of GTP failed to
block photoincorporation of [y32P]8-Azido GTP. These results
support the tentative finding seen in the initial attempt to

Figure 3-7 Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with increasing concentrations of
[y P]8-Azido GTP. Bovine 28S ribosomes (l jaM) were irradiated
with uv light for 10 min at 300 nW/cm on ice in the presence
of increasing concentrations of [v Pl8-Azido GTP (0.3 to 3
Results in the presence (B, D, & F) and absence (A, C,
of 10 nM GTP is also shown. The samples were irradiated
witn uv light for 10 min at 300 ^W/cm ónice and under dim
red light. The specific activity of the [y P]8-Azido GTP was
2.9 Ci/mmol just prior to use. Silver stained gels are on the
left and their corresponding autoradiograms on the right.
Autoradiograms were exposed for 7 days. The Mr markers used
were the same ones described in figure 3-2. See Methods for
additional experimental details.
i tn

67
Figure 3-7 A & B Two dimensional PAGE analysis of photoaffinity
labeled 28S ribosomes with 0.3 nM (9% saturation) [y P]8-Azido GTP
(panels A & B). The presence of 10 nM GTP (panel B) blocked
radiolabeling of S5.

68
c
Figure 3-7 C & D Two dimensional PAGE analysis of_ohotoaffinity
labeled 28S ribosomes with 1.0 nM (28% saturation) [y P]8-Azido GTP
(panels C & D). Note, that in the presence of 10 GTP (panel D)
$5 was not radiolabeled.

69
Figure 3-7 E & F Two dimensional PAGE analvsis of Dhntnaff-in-i+v
(panefs E8& Fl^Notl W1thht3‘-° *fJ56% saturation! [Y32P]8-Azido Gt{>
¿5 wfs not radiolabeled preSence of « mM gIp (panel F)

70
[r32p]8N3GTP CONCENTRATION (X106M)
Figure 3-7 G Densitometric analysis of the GTP specific and
nonspecific radiolabeling of ribosomal proteins S5 and S4
respectively. In panel G densitometric analysis of the
autoradiograms in panels A to F for the specific labeling of
S5 and nonspecific labeling of S4 is shown.

71
AR
GTP(lOjuM) - + - +
Figure 3-7 H Analysis of rRNA extracted from 28S ribosomes
irradiated in the presence of [y P]8-Azido GTP. This is an
analysis of the rRNA extracted from photoaffinity labeled 28S
ribosomes shown in figure 3-8, panels C & D ( the result in
all other panels was the same, no radiolabeling of rRNA). The
rRNA was extracted following uv irradiation by 9M urea and 3M
Li Cl and el ectrophoresed by ID urea PAGE (7.5% PAGE) as
described in Methods. The left panel is a methylene blue
stained gel (S) and its corresponding autoradiogram (AR) on
the right. The autoradiogram was exposed for 41 days. The
molecular weight markers are six synthetic poly(A)-tailed RNAs
ranging in length from 0.24 to 9.49 kb (Bethesda Research
Labs). No rRNA fragments were radiolabeled.

72
identify the GTP binding protein by 2D-PAGE analysis (figure
3-3), that S5 was specifically labeled by 8-Azido GTP. The
development of more stringent ionic and milder photoirradia¬
tion conditions did not eliminate nonspecific labeling of S4.
S4 was the most intensely labeled protein which increased
proportionally with the concentration of [y32P]8-Azido GTP.
S4 appears to be reciprocally labeled in the presence of
excess amounts of GTP (figure 3-7 G). The amount of S4
labeling increases in the presence of 10 *iM GTP, even though
the radionucleotide was diluted as much as 33 fold. This
suggests this site may be a lower affinity site for 8-Azido
GTP. No radiolabeling of 12S rRNA was detected (figure 3-7
H), suggesting no direct involvement of rRNA in binding of 8-
Azido GTP to 28S ribosomes.
Piscussion
Photoaffinity labeling of 28S ribosomes with 8-Azido GTP
revealed that a single 38 kDa ribosomal protein bound the GTP
analog in a specific manner (figure 3-2 to 3-7). Unlabeled
GTP could displace the radiolabeling of this protein
suggesting the site was specific for GTP. A 43 kDa protein
was also radiolabeled but the labeling was nonspecific because
excess amounts of GTP failed to block binding. The amount of
radiolabeling on the 38 kDa protein increased with increasing
concentrations of [a32P]8-Azido GTP and was saturable (figure
3-2 B) in a fashion consistent with the binding of this
compound to 28S ribosomes as determined by filter binding

73
assays (figure 2-3). The photolabeling reaction was done
under the same ionic conditions (reaction buffer B) as were
used in filter binding assays to assess [a32P]8-Azido GTP
binding to 28S ribosomes.
The identity of this labeled protein was examined by 2-
dimensional PAGE. Two proteins retained the label, one S4,
was the predominately radiolabeled protein but the labeling
was nonspecific because it was not diminished in the presence
of excess amounts of GTP. The second protein, S5, was
specifically radiolabeled by [a32P]8-Azido GTP. Excess
concentrations of GTP blocked binding to this protein
suggesting that it is the specific binding site for GTP.
Comparing these results to those of the 1-dimensional
analysis (figure 3-2), the 43 kDa nonspecifical ly labeled
protein correlates to S4 in both Mr and binding specificity,
and the 38 kDa protein would be S5 for the same reasons. The
only disparity was that the radiolabeling of S5 was less than
that seen on S4 in the two-dimensional PAGE analysis (figure
3-7). The disparity likely resides in differences in sample
preparation. In two-dimensional PAGE analysis, photolabeled
28S ribosomes are extracted under acidic conditions (pH 2.5)
to remove rRNA prior to separation in the first dimension in
urea also under acidic conditions (pH 4.3). Temple et al.
(1966) and Maliarik and Goldstein (1988) have observed that
some covalent bonds formed by the aryl (purine) nitrene
adducts are acid labile. In previous attempts to resolve

74
photolabeled ribosomes with the azido analog no radiolabeled
proteins were detected. Increasing the pH of the extraction
step to neutrality, only one radiolabeled protein, S4, was
found and it was not blocked in the presence of GTP (data not
shown). However when the pH of the first dimensional
separation was raised from 4.3 to 5.0 we were able to observe
not only the nonspecific labeling of S4 but also specific
labeling on S5 (figure 3-3 and 3-7). The separation of these
basic ribosomal proteins in the first dimension in urea could
not be done at any higher pH without risking alteration of the
relative mobilities of the proteins in a defined system
(Matthews et al.,1982). Never-the-less, under the elevated pH
conditions during sample preparation and analysis, it is clear
that S5 is the protein binding GTP in a specific manner.
Concerns about the survivability of the covalent bound formed
by the nitrene adduct, and photolabeling under more
physiological ionic conditions lead to studies to determine
the minimum photoirradiation required and optimal ionic
conditions for this reaction.
The minimum fluency (pW/cm2) and duration of irradiation
to achieve labeling of ribosomal proteins using 254 nm light
with 8-Azido GTP, were 300 pW/cm2 and 10 min, respectively
(figure 3-5). This was determined over the span of two
experiments in which both the [a32P] and [y32P]8-Azido GTP were
used, with no differences seen in the pattern of radiolabeled
proteins. O'Brien et al. (personal communication) found that

75
binding of [y32P]GTP to 28S ribosomes, under conditions similar
to those used here, resulted in no hydrolysis of the [y32P]
label even after several hours of incubation with the
ribosome. This indicates that under these binding conditions
[v32P]8-Azido GTP can be used with no hydrolysis of the [y32P]
to obscure the results of photoaffinity labeling.
The length of time required (10 min) for adequate levels
of radiolabeling are somewhat longer than reported by others
(typically 2 to 6 min at fluency < 1000 nW/cm2 on ice in
various buffer biological solutions, see Potter and Haley,
1983). However, irradiation for 4 min at 300 W/cm2 was
minimally acceptable in 1-dimensional systems, but due to
greater concentrations of protein (1 versus 0.5^M ribosomes)
it was not judged adequate for 2-dimensional analysis.
Several enzymatic systems which were subjected to
photoaffinity labeling saw no appreciable loss in function
when irradiated with 254 nm light at low temperatures for as
long as 10 min if the fluency remained below 800 W/cm2 (Potter
and Haley, 1983). These guidelines were used, since no
functional assay exist for the 28S ribosome which could be
used to monitor uv induced damage.
Geahlen and Haley (1977) reported that the half-life of
the 8-azido GMP (160 |iM at neutral pH) was approximately 20
sec in protein free buffer solution at approximately 1000
iiW/cm2 with 254 nm light. Since only minute quantities of the
8-azido GTP were available, it was not possible to examine the

76
uv degradation of the 8-azido to N2 and 8-NH2-GTP by
spectrophotometric means as done by Geahlen and Haley (1977).
Also, no suitable thin layer chromatography or other method
could be found that could discriminate between 8-azido and the
8-NH2 GTP though it was determined that the gamma phosphate
was not lost following irradiation under the conditions used
in this study (data not shown).
Efforts to develop more physiologically relevant ionic
conditions for GTP binding to 28S ribosomes focused upon two
components of the reaction buffer, MgCl2, and NH4C1. In the
initial binding experiments of this study the ionic
composition of the reaction buffer (B) was adopted from the
work of Bodley (Bacca, et al., 1976) who developed it to study
GTP binding to bacterial EF-G. The concentration of the
divalent cation, MgCl2 (20 mM), was reduced to 5 mM to
alleviate concerns about subunit dimerization which could
alter GTP binding to the ribosome. O'Brien (1971) showed that
the mitochondrial monoribosomes are stabilized at the higher
concentration of MgCl2 (20 mM MgCl2 in 100 mM KC1) or greater.
This occurs because divalent cations form salt bridges which
stabilize rRNA interactions between subunits which results in
subunit association or aggregation. At 5 mM MgCl2,
mitoribosomes subunits exist as discrete particles. GTP-Mg+2
binds as a complex to GTP-binding proteins. The Mg+2 ion binds
tightly and nM concentrations of the divalent cation commonly
suffice to fulfill this requirement (Gilman, 1987). Though

77
only modest increases in the photoincorporation of [y32P]8-
Azido GTP in 28S ribosomes were observed at 5 mM MgCl2
(figure 3-6), this reduced concentration of the divalent
cation was adopted.
The replacement of the monovalent cation NH4+ by K+ was
examined because NH4C1 is known to be toxic to the peptidyl
transferase activity of the mitoribosome large subunit
(Denslow and O’Brien, personal communication). Though we are
studying a nucleotide binding activity on the small subunit it
was deemed prudent to replace this monovalent cation in the
event NH4C1 is also toxic to some function of the small
subunit, and in the event that future studies of peptidyl
transferase activity might be undertaken in conjunction with
this GTP-binding activity. Additionally, we wanted to
increase the ionic strength to determine if GTP-binding would
be affected by increasing the stringency to more
physiologically relevant conditions. This was accomplished by
replacing NH4C1 with KC1 and testing the effect of higher
concentrations of KC1, equivalent to physiological
concentrations (70 to 100 mM). GTP-binding activity was
essentially unaltered at 100 mM KC1 when compared to binding
activity at 10 mM NH4C1 (table 3-1). This indicated that GTP
can bind the 28S ribosome even at moderate salt concentrations
suggesting the binding is not predominately electrostatic in
nature. Having established new ionic conditions (reaction
buffer A) photoaffinity labeling of 28S ribosomes by

78
[y32P]8-Azido GTP was examined again. Photoaffinity labeling
with increasing concentrations of 8-Azido GTP to a fixed
concentra-tion of the ribosome, resulted in radiolabeling of
the same proteins seen previously. S4 was nonspecifically
radiolabeled and S5 was specifically radiolabeled by [y32P]8-
Azido GTP. At very high saturating concentrations of the GTP
analog, other ribosomal proteins were also labeled in a
nonspecific manner (figure 3-7 E). At all concentrations of
[y32P] 8-Azi do GTP used, S5 radiolabeling was blocked in the
presence of excess GTP and the radiolabeling of S5 increased
with increased concentrations of the [y32P]8-Azido GTP.
Nonspecific radio-labeling of S4 also increased in a manner
comparable to that seen for S5 but interestingly S4
radiolabeling increased in the presence of excess GTP and
appeared to be saturable (figure 3-7 G). This suggested that
S4 bound some of the [y32P] 8-Azi do GTP displaced from the
binding site by GTP (10 nM) even though the specific activity
of the radiolabeled GTP analog was decreased by 33 to 3.3
fold. This suggests that S4 binds GTP in a manner different
than for S5, and is likely to have a lower affinity binding
site.

CHAPTER 4
PHOTOAFFINITY LABELING OF 28S RIBOSOMES BY [a32P]GTP
Introduction
Direct photoaffinity labeling of phosphofructokinase
(Ferguson, and Maclnnes, 1980) and tubulin (Nath, et al.,
1985; Hesse, et al., 1987;and Linse and Mandelkow, 1988) with
radiolabeled cAMP and GTP/GDP, respectively, encouraged us to
explore the use of the [a32P]GTP to photolabel the 28S ribo¬
some. The chemical structure at the site of covalent attach¬
ment for these purines is not known, but the fact that maximal
photolabeling of phosphofructokinase occurred at a wavelength
equivalent to its absorption maxima suggests that the adenine
portion of the nucleotide is the light sensitive chromophore
(Ferguson and Maclnnes, 1980). The use of the natural ligand
takes advantage of the increased binding affinity thereby
circumventing difficulties encountered due to lowered binding
affinities of the 8-Azido GTP analog for the 28S ribosome
(table 2-3). The disadvantages are that the mechanism of
photoactivation is unknown and therefore we do not know if
covalent bond formation is as nonselective a process as was
the case for the nitrene. Another disadvantage is that the
minimal quantum yield of product would be lower than for the
79

80
nitrene. In the case of cAMP, photolabeling of phosphofructo-
kinase was reported to be approximately 0.01% (Ferguson and
Maclnnes, 1980) several hundred times less than for the
nitrene. However that difficulty can be overcome to some
degree because [a32P]GTP can be obtained at specific activities
as much as 300 to 600 times greater than [a32P]- or [Y32P]8-
Azido GTP, respectively.
In this study we report successful photoaffinity labeling
of 28S ribosomes with [a32P]GTP. The photolabeling of ribo-
somal proteins was equivalent to that seen for [a32P]- and
[Y32P]8-Azido GTP, confirming that S5 is the specific site for
photoincorporation of GTP.
Materials and Methods
Reagents
[a32P]GTP, and ATP and [Y32P]ATP in triethylammoniurn salt
were purchased from Amersham at specific activities of 410 and
>3000 Ci/mmole, respectively. All unlabeled nucleotides were
purchased from Sigma Chemical Co. as Type I reagents as
described above.
Solutions
The same solutions as described above, in 8-Azido GTP
photoaffinity labeling experiments, were used in these
studies.

81
Methods
The same methods as described above, in 8-Azido GTP
photoaffinity labeling experiments, were used in these
studies.
Results
Photoaffinitv Labeling of 28S Ribosomes with lV2PlGTP
In an effort to substantiate the above findings
photolabeling of small subunits with [a32P]GTP was attempted.
Hesse et al. (1987) successfully photolabeled 6-tubulin with
[o32P]GTP suggesting the natural ligand could be used to
photoaffinity label the GTP binding ribosomal protein.
First, photolabeling conditions were varied to optimize
photolabeling. 28S Ribosomes (0.5 nM) were photoaffinity
labeled in presence of 0.5 nM [a32P]GTP, which corresponds to
80% saturation (calculation based on a Kd of 15.2 nM) of the
GTP binding site. Ribosomes were photolabeled for 2 and 10
min at increasing intensities (800, 1500, 2000, and 2500
nW/cm2) of uv light (254 nm) on ice and under dim room light.
The pattern of radiolabeling of 28S ribosomes by [a32P]GTP
(figure 4-1) was similar to that for [a32P]8-Azido GTP (figure
3-4) and [y32P]8-Azido GTP (figure 3-5). The 38 kDa ribosomal
protein was specifically labeled by GTP because excess amounts
of GTP blocked labeling of that protein. The 43 kDa ribosomal
protein was still nonspecifically labeled in the presence of
excess amounts of GTP as seen with the 8-Azido analog.

82
Ribosomes
+ ♦♦♦ + + ♦+ ♦♦+ + +«*■<••
G TP (5/xM)
Irrad. time (min) 2 10 2 2 2 5 10 200
Intensity (mW/cm2) 0.8 I.5 2.0H 2.5 1 0
Figure 4-1 A Optimization of the intensity and duration of uv
irradiation for photoaffinity labeling of 28S ribosomes with
[a„P]GTP. 28S ribosomes (0.5 ^M) were incubated with 0.5 uM
[a P18-Azido GTP (80% saturation) in reaction buffer B. The
samples were irradiated with uv light for 5 min at 800 nW/cm ,
on ice, prior to electrophoresis. [a PiGTP was diluted 10
fold with GTP to a specific activity of 32 Ci/mmol just prior
to use. In panel A is a representative lane of silver stained
(S) ribosomal proteins on the riqht and the correspondí’n
autoradiogram (AR) on the left. The AR was exposed for 24
before development. The presence and absence of ribosomes and
unlabeled GTP as well as the intensity and duration of uv (254
nm) irradiation are noted below the autoradiogram. The arrow
indicates the radiolabeled 38 kDa protein that is specifically
labeled by GTP.
8

83
[a32P] GTP
x
o
UJ
X
¡2
CL
UJ
>
UJ
m
210 2 2 2 5 10 20
0.8mW 1.5mW 2.0mW 2.5mW
Figure 4-1 B Densitometric analysis of the autoradiogram
shown in figure 4-1 A. Densitometric analysis was done for
the specifically labeled ribosomal protein (38 kDa) and the
nonspecifically labeled protein (43 kDa) ribosomal protein at
varying intensities and duration of uv irradiation. In the
presence (+) of 5 nM GTP, the 38 kDa protein was not
radiolabeled. The duration and intensity of uv irradiation is
indicated below the figure. For additional experimental
details see Methods.

84
Photolabeling of the 38 kDa protein by [a32P]GTP was more
dependent on the duration of irradiation than the intensity of
the uv light (figure 4-1 B). No differences were seen by
densitometric analysis in the amount of photolabeling of the
38 kDa protein when the intensity of uv irradiation was
increased from 800 to 2500 tiW/cm2 for 2 min. However the
amount of photolabeled 38 kDa protein was dramatically
increased nearly 10 fold when the duration of irradiation was
lengthened to 10 min at 2500 jiW/cm2. The amount of
photolabeling at 800 and 2500 jiW/cm2 was in fact nearly
identical for irradiation at 10 min. This data suggests that
irradiation for 5 min at 800 nW/cm2 would produce equivalent
amounts of radiolabeling to that seen for 5 min at 2500 nW/cm2
(figure 4-1 B). The amount of radiolabeling in that case was
sufficient for photoaffinity labeling of 28S ribosomes so uv
(254 nm) irradiation at 800 ^W/cm2 for 5 min was adopted for
subsequent photolabeling experiments with [a32P]GTP.
To determine the extent of binding specificity of the 28S
ribosome several nucleotide tri- and diphosphates were tested
for their ability to block photoaffinity labeling of 28S
ribosomes by [a32P]GTP. Of the nucleotides tested, only GTP
and GDP, and to a much lesser extent, GMP, were able to block
photolabeling of the 38 kDa ribosomal protein by [a32P]GTP
(figure 4-2 A). These findings agree with the results of
filter binding assays (figure 2-2) and photoaffinity labeling
experiments with 8-Azido GTP, that there is a single GTP

Figure 4-2 A Specificity of tie photoinduced radiolabeling of
28s ribosomal proteins by UP] GTP in the presence of other
nucleotides. Ribosomes small subunits (0.5 ^M) were incubated
in the presence of 0.28 nM [a P]GTP (50% saturation) in the
presence (+)and absence (-) of 5 unlabeled nucleotide (GTP,
GDP, GMP, ATP, CTP, UTP, and GTP + ATP). The samples were
irradiated with uv light for 5 min at 800 tiW/cm on ice, in
reaction buffer A. In panel A a representative silver stained
lane of 28S ribosomes is shown on the left and the
corresponding autoradiogram (AR) on the right. The presence
and absence of ribosomes, uv irradiation, [a P]GTP, and
unlabeled nucleotide is shown under the AR. The specific
activity of the [a PlGTP was 410 Ci/mmol and the autoradiogram
was exposed for 24 nours. The arrow identifies the 38 kDa
protein specifically radiolabeled by GTP.

86
S AR
Ribosomes -++++ + + + ++ +
UV ++-+ + ++ + + + +
[«32p1gtp +-+ + ++++ + + +
GTP +
GDP +
GMP - +
ATP +
CTP +
UTP + -
GTP+ATP +

87
x
o
Lü
X
¡2¡
X
Lü
>
s
Lü
Od
100 r
80 -
60
40
20
0
E53 38 kDa
di 43 kDa
rexTl
- +
NXP GTP
+
GDP
+ +
GMP ATP
+ +
CTP UTP
+
GTP
ATP
Figure 4-2 B Densitometric analysis of the autoradiogram
(panel A) of 28S ribosomes photoaffinity radiolabeled with
[a P]GTP in the presence (+) and absence (-) of various
nucleotides. The analysis was done for both the specificall
radiolabeled 38 kDa and the nonspecifically radiolabeled 4
kDa protein. In the presence of 5 GTP and GDP photoinduced
radiolabeling of the 38 kDa protein by [a P]GTP was blocked,
and 5 nM ATP blocked radiolabeling of the 43 kDa protein. See
Methods for additional experimental details.

88
binding site on the 28S ribosome that is specific for GTP and
GDP. The site for nucleotide binding is a 38 kDa ribosomal
protein, identified as S5 by 2-dimensional analysis of 28S
ribosomes photoaffinity labeled with 8-Azido GTP (figure 3-3
and 3-7).
The 43 kDa ribosomal protein was radiolabeled by [a32P]
GTP, except in the presence of ATP (figure 4-2 A). Densito-
metric analysis (figure 4-2 B) indicated that labeling of the
43 kDa protein was completely blocked in the presence of
excess amounts of ATP suggesting for the first time that this
site may specifically bind a nucleotide.
Mi 11ipore filter binding and binding inhibition assays
(O'Brien et al., 1990; and figures 2-1 and 2-2) strongly
indicated that a high affinity site that binds one molecule of
GTP or GDP exists on the 28S ribosome. Autoradiographic
analysis of one dimensional gels following photoaffinity
labeling of 28S ribosomes suggest that a 38 kDa ribosomal
protein is the specific GTP/GDP binding site.
Photoaffinity labeling of this site results in a small
but reproducible percentage of the site covalently labeled by
[a32P]GTP. Therefore analysis of the amount of radiolabeling
of the 38 kDa ribosomal protein following photolabeling of 28S
ribosomes with increasing concentrations of [a32P]GTP would
produce a binding curve similar to that seen in filter binding
assays with intact small subunits if they represent the same
binding activity.

89
Small subunits (0.5 |aM) of mitochondrial ribosomes were
photoaffinity labeled with increasing concentrations (0.05 to
1.5 nM) of [cc32P]GTP and in the absence and presence of 5 nM
GTP in reaction buffer A as described above. Photolabeling
was done at 800nW/cm2 for 5 min on ice under dim room light.
Following autoradiographic analysis (figure 4-3 A) of
ribosomal proteins resolved on a 12% polyacrylamide Laemmli
gel, the radiolabeled 38 and 43 kDa protein bands were excised
and analyzed for the amount of bound [a32P]GTP by 6-
scintillation counting (figure 4-3 B, upper panel). The
amount of radioactivity due to specific radiolabeling of the
GTP binding site was determined by the difference in cpm found
on the 38 kDa protein in the presence and absence of 5 nM GTP.
This net radioactivity was converted to pmol of bound [a32P]GTP
based upon the calculated percent photoincorporation of the
labeled GTP and its specific activity as discussed below.
The pattern of radiolabeling was identical to previous
studies.
The amount of radiolabeling the 38 kDa protein did
increase rapidly with increasing concentrations of [a32P]GTP.
At molar ratios of [a32P]GTP to ribosomes greater than one, the
radiolabeling of the 38 kDa protein did not increase further
indicating saturation of the GTP binding site. Assuming that
at saturation one molecule of GTP was bound to a ribosome the
percent of photoincorporation of [a32P]GTP into the 38 kDa
protein was determined. At a specific activity of 116.4

Fiaure 4-3 A & B Determination of the binding affinity of
[a PlGTP for the specifically labeled 38 kDa ribosomal protein
by pnotoaffinity labeling of 28S ribosomes. 28S Ribosomes
(0.5 nM) were incubated in reaction buffer A with increasing
concentrations (0.05, 0.13, 0.4, 0.6, 0.9, and 1.5 jiM) of
[a P]GTP (corresponding to 10, 25, 70, 90, 120, and 200%
saturation of the GTP binding site on the 28S ribosome) and in
the presence (+) and absence (-) of 5 *iM GTP. Samples were
irradiated as described in figures 4-1 and 4-2. The specific
activity of [aP]GTP was 278 Ci/mmol. In panel A a
representative lane of silver stained (S) ribosomal proteins
is shown on the left and the corresponding autoradiogram (AR)
on the right. The concentration of the [a P]GTP and GTP shown
below the autoradiogram. The arrow identifies the 38 kDa
protein specifically radiolabeled bv GTP. In panel B the
amount of radioactivity found in the 38 kDa protein bands due
to specific photolabeling of that protein was plotted versus
the concentration of ligand added. The amount of specific
radiolabeling of this protein was found by subtracting the
radioactivity found in the 38 kDa gel plug in the presence of
5 nM GTP from that amount found in the same protein in the
absence of 5 nM GTP. Additionally the amount of radioactivity
found in 38 kDa protein due to dilution of [a P]GTP with GTP
was also subtracted. The amount of [a P]GTP covalently bound
to the 38 kDa protein was calculated based on the specific
activity and percent photoincorporation as described in
Results. Additionally, the amount of radiolabeling of the
nonspecifically radiolabeled 43 kDa protein is shown. The
amount of radioactivity found in the 43 kDa band in the
absence of 5 jiM GTP is plotted versus the concentration of
[a^PlGTP. The 43 kDa band was not specifically labeled by
[aPJGTP therefore the amount of radioactivity found in the
corresponding band in the presence of 5 nM GTP was not
subtracted. However, the amount of [aP]GTP bound to the 43
kDa band was calculated as above, based on the same specific
activity and percent photoincorporation as used for the 38 kDa
protein.

CPM
91
A
s
GTP (5mM) - - + - +
B
GTP ADDED (X 106M)

92
0.0 0.2 0.4 0.6 0.8 1.0
FREE GTP (x 106M)
Figure 4-3 C Comparison of binding curves for photoinduced
covalent binding of [aP]GTP to the 38 kDa ribosomal protein
versus noncovalent binding to 28S ribosomes as determined by
filter binding assays. The concentration of free [a P]GTP
(calculated from data in panel B) was plotted versus that
covalently bound to the ribosome in panel B. The binding of
covalently bound [aP]GTP to th£ 38 kDa protein was compared
to noncovalent binding of [aP]GTP to 28S ribosomes as
determined by filter binding assays. The same preparation of
ribosomes was used in both experiments. Scatchard analysis of
both binding isotherms revealed apparent dissociation
constants of 9.8 ± 2.4 and 42.6 ± 23.2 nM for covalent and
noncovalently bound GTP, respectively. The number of binding
sites for GTP on the ribosome for both experiments was 1.0.

93
Ci/mmole (or 225 cpm/fmol, at the time of analysis), 0.5 /mol
of [a32P]GTP was bound to 25 pmol of ribosomes when the binding
site was saturated, for a percent photoincorporation of 2 X
10'3%. The amount of covalently bound [a32P]GTP was converted
to concentrations of bound versus free ligand based upon the
above specific activity and percent photoincorporation for
[a32P]GTP. Scatchard analysis of the resulting binding curve
yielded an apparent dissociation constant and number of
binding sites on the 28S ribosome of 42.6 ± 23.2 nM (kD ± SE)
and 1.0, respectively (figure 4-3 C). This value is not
significantly different from that obtained by filter binding
assays (15.2 ± 8.64 nM, table 2-3) where radiolabeled GTP was
bound to small mitochondrial ribosomal subunits. This
suggests that the specific GTP binding site is the 38 kDa
protein.
The radiolabeling of the 43 kDa protein with [a32P]GTP was
not blocked in the presence excess amounts of GTP. However
radiolabeling of this protein did increase slowly as the
concentration of [a32P]GTP increased. The radiolabeling of the
43 kDa increased in a nearly linear manner suggesting a much
lower affinity for [a32P]8-Azido GTP for this protein.
Two dimensional PAGE analysis of 28S ribosomes
photoaffinity labeled with [a32P]GTP in the presence and
absence of GTP and ATP was done to confirm the identity of the
GTP binding protein. 28S Ribosomes (1.0 jiM) were photo¬
affinity labeled (800nW/cm2 for 5 min on ice) with 0.53 nM

Figure 4-4 Two dimensional PAGE analysis of 28S ribosomes
photoaffinity labeled with [a P]GTP. Bovine 28S ribosomes (1
nM) were irradiated in the presence of 0.53 *iM [a P]GTP (50%
saturation, all panels) and 10 jiM GTP (panel B), or 10 yM ATP
and GTP (panel D). The samples were irradiated with uv light
for 5 min at 800 tiW/cm on,, ice and under dim light. The
specific activity of the [« P]GTP was 336 Ci/mmol. Silver
stained gels (S) are on the left and their corresponding
autoradiograms (AR) on the right. Autoradiograms were exposed
for 4 days. The molecular weight markers used were the same
ones described in figure 3-2. See Methods for additional
experimental details.

95
A
S
A R
_ S4
S*J
\
-/*
^ .
/
S5 * A
S5
. t
X*
b
Figure 4-4 A & B Two dimensional
ribosomes photoaffinity la
incubated and uv-i madia-
(panel A) and presence
presence of GTP radiolabe'
PAGE analysis of 28S
beled with IV P]GTP. Ribosomes were
[« 2P] <
ed with [cVPJGTP in the
panel B) of 10 *iM GTP.
ing of S5 was blocked
absence
In the

96
Figure 4-4 C & D Two dimensional PAGE analysis of 28S
ribosomes photoaffinity labeled with [a PlGTP. Ribosomes were
incubated and uv-irradiated with [a P]GTP in the presence of
10 ATP alone (panel C) and presence of 10 uM ATP and GTP
(panel D). In tne presence of ATP radiolabeling of S4 was
blocked and with the addition of GTP, radiolabeling of S5 was
also blocked.

RELATIVE PEAK HEIGHT
97
-ATP +ATP
Figure 4-4 E Densitometric analysis of the 2D-PAGE
autoradiograms (panels A to D) for the specific labeling of S5
by GTP and nonspecific labeling of S4. The presence (+) or
absence (-) of 10 GTP or ATP is indicated below the figure.
GTP blocked photoinduced radiolabeling of S5 while ATP blocked
radiolabeling of S4.

98
GTP(IOp.M) + ~ + - +- + ~
ATP(IOfiM) -- + + — + +
Treatment A B C D
AB C D
Figure 4-4 F Autoradiographic analysis of rRNA extracted from
285 ribosomes irradiated in the presence of [aP]GTP. In
panel F, an analysis of the rRNA extracted from photoaffinity
labeled 28S ribosomes shown in panels A to D. Following uv
irradiation the rRNA was extracted in 9M urea and 3M Li Cl and
electrophoresed by ID urea PAGE as described in Methods. The
left panel is a methylene blue stained gel (S) and its
corresponding autoradiogram (AR) on the right. The
autoradiogram was exposed for 30 days. The molecular weight
markers are six synthetic poly(A)-tailed RNAs ranging in
length from 0.24 to 9.49 kb (BRL). No rRNA fragments were
radiolabeled.

99
[a32P]GTP in reaction buffer A as described above. In the
absence of 10 nM GTP, S5 was specifically radiolabeled with
[a32P]GTP (figure 4-4) as was the case for [a32P]8-Azido GTP
(figure 3-3) and [y32P]8-Azido GTP (figure 3-7) but the
majority of the radiolabeling was still on the nonspecifically
labeled ribosomal protein, S4 as seen before. However in the
presence of 10 *iM ATP, radiolabeling of S4 was blocked
completely, and the specific radiolabeling of S5 was more
clearly visualized (figure 4-4 C and D). Radiolabeling of S5
by [a32P]GTP was completely blocked in the presence of 10 nM
GTP (figure 4-4 B, D and E) as seen in all previous studies
with [a32P] and [y32P]8-Azido GTP. This suggests strongly that
the site of the specific and high affinity GTP binding
activity observed on the small subunit of the mitochondrial
ribosome is on ribosomal protein, S5. Autoradiographic
analysis of 12S rRNA suggested no photolabeling occurred
(figure 4-4 F)
Continued Radiolabelina Studies on S4
In an earlier experiment an interesting observation was
made. In the absence of uv irradiation [a32P]ATP radiolabeled
the 43 kDa (S4) ribosomal protein (data not shown) for a
particular preparation of 28S ribosomes. In figure 4-5
radiolabeling of 28S ribosomes in the presence and absence of
uv (254 nm) irradiation was investigated. Two different
preparations of 28S ribosomes (0.5 nM) were incubated with

Figure 4-5 A Determination of the requirement for uv
irradiation to radiolabel 28S ribosomes with UP]ATP and
[a P]GTP. Duplicate samples of 28S ribosomes (0.5 uMl were
incubated in reaction buffer A with either 0.26 nM [a PlATP
or [a P]GTP (50% saturation, based on an apparent Kd of 15.2
nM). One sample was irradiated with uv light for 10 min with
800 jiW/cm on ice as described in Methods and the other
incubated on ice under dim light with no irradiation. One
ribosomal preparation (#10221 was .used for both NTPs and a
second preparation (#1028) for [aP]GTP only. The silver
stained (S) gels are on the left of their corresponding
autoradiograms (AR). The molecular weight markers are the
same shown in figures 3-2. The irradiation with uv light,
[a P]NTP, and ribosome preparation used are indicated below
figure. The specific activity of both [a P]NTPs was 122
Ci/mmol. The autoradiograms were exposed for 4 days. The
arrow denotes the location of S5 (38 kDa).

101

RELATIVE PEAK HEIGHT
102
[a32P] GTP [a32P] ATP
Figure 4-5 B Densitometric analysis of 28S ribosomes
incubated in the presence of U P]ATP and [a P]GTP in the
presence (+) and absence (-) of uv irradiation. The
autoradiogram shown in panel A was analyzed by densitometry
and shown in panel B. The irradiation of the samples with uv
light, [a PjNTP, and ribosome preparation used is indicated
below the figure. For additional experimental details refer
to Methods.

103
either 0.265 nM [a32P]GTP or [cc32P]ATP in reaction buffer A and
half the samples were irradiated with 800 nW/cm2 uv light for
10 min on ice and under dim room light. The concentration of
[a32P]GTP used corresponds to 50% binding (based on a Kd of
15.2 nM for GTP) of the GTP binding site on the ribosome.
Ribosomal preparation #1022, S4 was radiolabeled by both
[a32P]GTP and [a32P]ATP even in the absence of uv irradiation
(figure 4-5 A, see AR lanes for rib. prep. #1022). In the
other ribosomal preparation (#1028) radiolabeling of S4 was
not detected (fig 4-5 A, see last two AR lanes on far right of
figure) . Silver staining of the two ribosomal preparations
resolved on a Laemmli gel indicated that S4 was absent in
second preparation (#1028) likely accounting for this
discrepancy (figure 4-5 A, S lanes). Ribosomal protein S5 was
not radiolabeled by [a32P]GTP or [a32P]ATP in either ribosomal
preparation in the absence of uv irradiation. S5 was only
radiolabeled by [a32P]GTP in the presence of uv irradiation
(figure 4-5 A and B). The radiolabeling of S4 in the absence
of uv irradiation not only survived boiling for 4 min in
Laemmli sample buffer which contained 2% SDS but also
resolution on a reducing Laemmli SDS polyacrylamide gel.
Clearly radiolabeling of S4 by [a32P]GTP or ATP occurred by a
different mechanism than the photolabeling of S5 by [a32P]GTP.
To determine if the uv independent radiolabeling of S4 by
[a32P]ATP has properties of a enzymatic reaction suggesting a
catalytic site on the ribosome a time course (figure 4-6) for

104
S AR
94 -
67 — ;?
43 -
30 -
20.1-
14.4-
Temp.
• •
• •
Time (min)
ATP Í a or y 32 pl
0.2 2 5 15 30 60
a
30 30
a y
Figure 4-6_A Time course for the uv independent radiolabeling of
S4 with [a P] and [y P]ATP. Duplicate samples of 28S ribosomes
(0.5 nM) were incubated in reaction buffer A with 1.0 nM [a P] for
varying times (0.2 to 60 min) and 1.0 nM [y P]ATP for 30 min. One
of the duplicate samples was incubated on ice (0°C) and the other
at 37°C under dim light. In panel A a representative silver
stained (S) gel is shown on the left and the corresponding
autoradiogram (AR) on the right. On the far right a AR comparing
radiolabeling of ribosomes with [a P] and [y P]ATP is shown. The
arrow indicates the presence of S4 protein. The [aP] and [y P]ATP
were diluted 10 and 100 fold with ATP to a specific activity of 30
and approximately 70 Ci/mmol respectively. The arrow identifies S4
protein (43 kDa). The autoradiogram was exposed for 45 min.

105
x
o
Lü
X
¡2¡
CL
Lü
>
5
Lü
CU
Figure 4-6 B Densitometric analysis for the radiolabeling of
S4 by [a P]ATP at 0°C and 37°C over increasing length of time.
Densitometric analysis was performed on the autoradiogram in
figure 4-6 A. Refer to Methods for additional experimental
detai 1s.

106
the radiolabeling of 28S ribosomes by [a32P]ATP was done at 37°
and 0°C. Small subunit ribosomes (0.5^M) were incubated with
either luM [a32P] or [y32P]ATP diluted with unlabeled ATP
reaction buffer A for an increasing period of time at 0°C or
37°C. Radiolabeling did increase with time at 0°C leveling
off after 30 min but almost no labeling occurred at physio¬
logical temperature (figure 4-6 A and B). This suggested that
the radiolabeling of S4 by [ nature. Additionally, comparison of the radiolabeling
patterns (figure 4-6 A right hand panel) of [a32P]ATP and
[y32P]ATP were identical thereby not revealing any autophos¬
phorylation of 28S ribosomes by [y32P]ATP.
To summarize the finding for the radiolabeling of S4 it
was found that [a32P]ATP failed to bind to 28S ribosomes in a
comparable manner as did [a32P]GTP as analyzed by filter
binding assays (figure 2-1). However [a32P]ATP did appear to
bind to a fractional site on the ribosome (n=.09) with an
apparent Kd of 77 nM (table 2-1). Both [a32P]GTP and ATP
radiolabel S4 independent of uv irradiation and that this
activity was not found in all preparations of ribosomes
(figure 4-5 A and B). ATP was the only nucleotide that could
block radiolabeling of 28S ribosomes by both [a32P]ATP and
[a32P] GTP (figures 4-2, and unpublished data). The
radiolabeling of [a32P]ATP occurs at 0°C and not at
physiological temperatures and is time dependent (figure 4-6).

107
Discussion
The natural ligand GTP and its photoreactive analog, 8-
Azido GTP bind specifically to S5. Binding to this protein
was revealed by autoradiographic analysis of one and two-
dimensional polyacrylamide gels following photoaffinity label¬
ing of 28S ribosomes. The photolabeling of S5 by [a32P]GTP
could be blocked effectively only with GTP and GDP which
agrees not only with the results of filter binding inhibition
assays (figure 2-2) but also with photolabeling experiments
using radiolabeled 8-Azido GTP (figure 3-3 and 3-7).
Following photoaffinity labeling a very small percentage
(< 0.1%) of GTP or 8-Azido GTP remains covalently bound to the
ribosomal protein. The addition of three negative charges
(from the triphosphate group of the radioligand) to an average
protein of 40 kDa mass is not expected to have much of an
effect on the electrophoretic mobility of the protein in the
two dimensional electrophoretic system used in these
experiments (Matthews, et al., 1982). Superimposition of the
autoradiogram with the dried two-dimensional gel revealed the
radiolabeled protein to be S5. Most of the individual
ribosomal proteins have distinctive shapes, which aids in the
identification of radiolabeled proteins, especially if the
mobility of the labeled protein differs somewhat from that of
the unlabeled protein. The radiolabeled image of S5 matched
precisely the corresponding silver stained protein spot on the
two-dimensional polyacrylamide gel (figures 3-3, 3-7, and

108
4-4). This suggests strongly that S5 with the covalently
bound nucleoside triphosphate migrates in the same manner as
S5. The additional three negative charges did not retard the
migration of S5 in the first dimensional separation to any
detectable degree, since the separation of proteins in this
first dimension is not as sensitive to alterations in charge,
as in isoelectric focusing. Moreover, other labeling studies
with mammalian mitochondrial ribosomes, such as iodination
(Denslow and O'Brien, 1984) and affinity labeling with
chloramphenicol analogs (Harville and O’Brien, unpublished
data), indicated the labeling did not affect the mobility of
ribosomal proteins in this electrophoretic system (Matthews,
et al., 1982).
Photolabeling of S5 was saturable and quantification of
the covalently bound [a32P]GTP revealed the radiolabeling
reaction to have the same concentration dependence as GTP
binding to ribosome subunits determined in the filter binding
assays (figure 4-3). This suggested that specific binding of
GTP to the 28S ribosome was due to binding to S5.
The nonspecific labeling of S4 was independent of uv
irradiation. It survived not only strong denaturing
conditions, such as boiling in SDS and migration in SDS
containing polyacrylamide gels, but also extraction in 10 M
urea and 3 M Li Cl for 2-dimensional PAGE analysis.
Radiolabeling of S5 requires uv irradiation suggesting
strongly these two proteins bind GTP by different mechanisms.

109
Radiolabeling of S4 also had a curious pattern. In the
presence of excess amounts of GTP, which would block
radiolabeling of S5, radiolabeling of S4 would not only not be
blocked, but would increase in an apparently reciprocal
manner. It was apparent that some of the radio-labeled GTP or
even its 8-Azido analog that was displaced by GTP now bound to
S4. The increase in radiolabeling of S4 even when the
specific activity [a32P] was diluted, by the addition of excess
amounts of GTP, was unexpected. One explanation would be that
S4 has a low affinity for GTP. Protein S4 does not bind GTP
specifically and binds [a32P]ATP as well. In fact ATP is the
only nucleotide found that blocks radiolabeling of S4 by
[a32P]GTP or [a32P]ATP. This finding was used in 2-dimensional
analysis of photoaffinity labeling of 28S ribosomes by
[a32P]GTP to block labeling on S4 to more clearly demonstrate
specific labeling of S5 (figure 4-4).
Scatchard analysis of GTP binding to ribosomes by filter
binding assays revealed only one high affinity binding site
for GTP on the ribosome with no indication that another low
affinity site existed. This is likely due to the fact that
these assays cannot measure binding with dissociation
constants in the high range or higher. This is due to
washing steps, used in filter binding assays to separate bound
from free ligand. Ligands that bind loosely have higher off
rates thereby are more susceptible to loses during washing.
An alternate method that may reveal a putative second lower

no
affinity site would be equilibrium dialysis however we lacked
sufficient quantities of ribosomes required for this analysis.
However quantitative analysis of the amount of [a32P]GTP
covalently bound to S5 and S4 in photolabeling experiments,
was done and the shape of the binding curves do reveal
differences in binding affinity (figure 4-3 B). The curve for
S5 was very steep and hyperbolic. Scatchard analysis of the
covalently bound [a32P]GTP revealed a high affinity binding
activity equivalent to that seen in filter binding assays.
This suggested that S5 was the high affinity binding site for
GTP on the ribosome. This calculation was based upon the
assumption that at saturation, one molecule of GTP was bound
to the ribosome as determined by filter binding assays. The
binding curve for GTP to S4 (figure 4-3 B) was nearly linear
and did not increase as rapidly as for S5 suggesting a low
affinity by [a32P]GTP for this protein. A dissociation
constant could not be calculated because saturation was not
demonstrated and the number of binding sites for GTP to S4 was
not known.
The nature of the binding of S4 to ATP or GTP is also un¬
known. Since irradiation by uv was not required (figure 4-5)
the binding occurs by a different mechanism than seen for S5.
The binding to S4 was resistant to moderate salt concentration
and was enhanced at low temperatures ( 0°C) versus
physiological (37°) temperatures suggesting the binding is
more hydrophobic in nature than electrostatic. The enhance-

Ill
ment in binding at low temperatures also suggests no enzymatic
action such as seen in ADP ribosylation. Ribosylation of the
ribosome can be ruled out not only because of the low tempera¬
ture but because the label resided in the a32P and not in the
ribose and no NADP was added to the reaction. Additionally
phosphorylation was ruled out because [a32P]GTP was used so no
label resided on the y-phosphate. Additionally, when [y32P]GTP
was used no differences were seen in radiolabeling patterns
between the two isotopes (figure 4-6).
The uv independent binding was able to survive harsh
denaturing conditions such as boiling in SDS and migration in
SDS containing polyacrylamide gels. The reason for such
binding behavior is not readily apparent.

CHAPTER 5
ISOLATION, IDENTIFICATION, AND PARTIAL AMINO ACID SEQUENCE
OF S5 AND OTHER RIBOSOMAL PROTEINS
Introduction
Separating ribosomal proteins from any source has always
been a formidable task due to their similarities in molecular
mass (6 to 60 kDa), large number of proteins in the various
subunits (especially in the mi toribosome, see Table 1-1),
isoelectric point (8 to 12), and in some cases extreme
hydrophobicity (Kaltschmidt, 1971; and Matthews et al., 1982).
The classical methods, ion exchange and size exclusion
chromatography are quite laborious, provided inadequate
resolution of individual proteins and suffered from low
recovery of protein. Recently reverse phase high performance
liquid chromatography (RP-HPLC) has become the method of
choice. Kerlavage et al.(1983) resolved nearly all of the
ribosomal proteins from all subunits in E. coli. Hi rano et
al. (1987) also separated ribosomal proteins from B.
stearothermophi 1 us and obtained amino-terminal sequence
information from several of the proteins. These separations
were done on a wide pore reverse phase alkyl si lane columns of
varying carbon chain lengths, with an increasing gradient of
organic solvent in the presence of triflouroacetic acid (TFA).
112

113
The majority of these bacterial proteins separated as pure
proteins. Three properties of this separation method that
were most responsible for the high degree of resolution were:
the requirement for a modifier, TFA, presumably to act as a
counter-ion; the advent of wide pore (300A) silica beads to
allow passage of larger molecules; and the high resolving
capabilities of HPLC columns with theoretical plate numbers of
>10,000. The use of RP-HPLC with organic solvents,
acetonitrile or 2-propanol, and TFA as a modifier has gained
widespread use in separating not only ribosomal proteins but
other proteins as well (Kamp et al.,1984; and Kamp, 1986).
A variation of the above method has proven useful when
one deals limiting amounts of protein, narrowbore RP-HPLC.
Narrow bore RP-HPLC was used extensively in this study to
isolate ribosomal proteins to purity in order to conserve
ribosomal protein as well as other reagents. The method used
is described below in Methods, and simply utilized a column
with a reduced inner diameter (2.1 mm). Narrow bore columns
are operated at linear flow velocities equivalent to
conventional bore columns (4.6mm, I.D.) columns by decreasing
the flow rate to the square of the reduction in column
diameter as shown in the equation:
Flow a = (ra / rb)2 (Flow b)
For example, an equivalent flow rate for a narrow bore (rb) in
comparison to a conventional (ra) HPLC chromatographic
separation at a flow rate of 1 ml/min would be 0.047 ml/min.

114
Also assuming the column efficiency is nearly the same and the
column capacity is not exceeded the same amount of protein
would be eluted into a smaller volume of solvent thereby
increasing the sensitivity by about 7 to 10 fold. The
conservation of protein and smaller volumes was an asset in
obtaining amino acid sequence information by micro gas-phase
sequencing methods employed in this study.
In some cases ribosomal proteins were not obtained in
sufficient purity in the RP-HPLC eluant or for other reasons
were digested into peptides. These were further resolved on
one-dimensional Laemmli gels and electroblotted onto a solid
phase support for sequence analysis. Western blotting of
proteins from PAGE gels onto TFA derivatized glass fiber
sheets was initially described by Aebersold et al. (1986) and
Vandekerckhove et al.(1985). However with the use of glass
fiber supports initial and repetitive cycle yields often
decreased with decreasing amounts of protein and small
quantities (<100 pmol) proved difficult to sequence (Yuen et
al., 1986). The search for a superior blotting support has
led to the adoption of PVDF which has proven especially useful
for electroblotting of hydrophobic proteins and peptides
(Pluskal et al., 1986, and Matsudaira, 1987).
I report here the separation of 28S ribosomal proteins
and the ribosomal identity of most of the eluted proteins.
Most of the identities were determined by coelectrophoresis
of eluted proteins with a small amount of 28S proteins on

115
two-dimensional gels. I also will show S5, the GTP-binding
protein, elutes as a pure protein and identified not only by
the above method but also by immunoblot analysis with a anti
S5 monospecific antibody. The amino-terminal sequence of a
number of 28S ribosomal proteins as well as considerable amino
acid sequence derived from peptides of S5 will also be shown.
Homologies in the amino acid sequence found in peptides of S5
to conserved regions in other GTP-binding regions involving
not only the triphosphate chain but also the guanine base will
be discussed.
Materials and Methods
RP-HPLC Mobile Phase Solutions
Aqueous Mobile Phase: HPLC grade water (Fisher Scientific)
with 0.1% trifluoroacetic acid (TFA, sequencer grade)
Organic Mobile Phase: HPLC grade 2-propanol (Fisher
Scientific) with 0.1% TFA
Electroblottina Solutions
Blotting Buffer: 0.25 M Tris-HCl, pH 8.3, 20% HPLC grade
methanol (Fisher Scientific), 192 mM glycine, and 0.01%
SDS (ultrapure grade)
Destain Solution: 50% HPLC grade methanol, and 2% HPLC grade
acetic acid
Immunoblot Solutions
Blocking Solution: 3% Bovine Serum (v/v), 150 mM NaCl, lOmM
Tris-HCl, pH 7.5, and 0.01% sodium azide

116
Incubation Buffer: 3% Bovine Serum (v/v), 150 mM NaCl, 10 mM
Tris-HCl, pH 7.5, 0.01% sodium azide, 0.5% Triton X-100,
and 0.01% SDS
TBS: 10 mM Tris-HCl, pH 7.5, and 150 mM NaCl
TBSA: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.01% Na-azide
Column Elution Buffer: 0.5 M HPLC grade acetic acid, and 1%
dioxane
Eauipment
1) High Pressure Liquid Chromatograph: Hewlett Packard model
#1090A with integral photo-diode array detector. The detector
has a Hewlett Packard model# 9121A double 3.5 in disk drive,
model# 65 personal computer, and a model# 7470 plotter device.
The diode array detector was capable of storing up to 8
distinct wavelengths simultaneously and collecting spectral
information as desired. One signal was split (signal 1, at 214
nm) to be analyzed by a Hewlett Packard model# 3390A
integrator. The chromatogram had a built in column oven to
maintain a constant solvent and column temperature.
2) Gas Phase Amino Acid Microsequencer: Applied Biosystems
(ABI, Foster City, CA) model# 470A with on-line ABI model#
120A PTH amino acid analyzer. All solvents used in
conjunction with this instrument were purchased from Applied
Biosystems Incorporated and were of Sequencer grade.
3) Savant Speed Vac concentrator, model# SVC-100H
4) IEC lab top general purpose centrifuge
5) Beckman table top high speed microcentrifuge

117
6) 96 well dot blot apparatus
7) Electroblotting device, Bio-Rad model# transblot
Extraction of Ribosomal Proteins for RP-HPLC analysis
Ribosomes were extracted in Mg(Ac)2-g 1 acial acetic acid
to remove rRNA by a modified procedure of Hardy et al. (1969)
prior to RP-HPLC analysis. To 28S or 39S ribosomes 1 M
Mg(Ac)2 was added to a final concentration of 32.2 mM and
allowed to stir for 5 min at 4°C. Concentrated HPLC grade
glacial acetic acid was then added to a final concentration of
66% of the total volume. The mixture was stirred at 4°C for
1 h, then centrifuged at 140,000 x g for 30 min at 4°C. The
rRNA pellet was re-extracted as described above for 20 min and
the supernatants pooled and dialyzed against 6% HPLC grade
acetic acid for 2 h prior to RP-HPLC analysis.
Reverse Phase HPLC of Ribosomal Proteins
Purification of mitochondrial ribosomal proteins for
chemical microsequencing purposes was accomplished by RP-HPLC.
Chromatographic separations were accomplished with a Hewlett
Packard 1090A HPLC with a photodiode array detector. This
detector allowed multiple wavelengths to be monitored during
an analysis. This capability allowed for the subtraction of
a reference signal of a wavelength (540 nm) from the signal of
interest (219, and 280 nm) which resulted in a chromatogram
largely devoid of baseline drift due to changes in solvent
composition during gradient runs. Spectral information for
all peaks were also stored at a 4 nm intervals between 210 and

118
450 nm for subsequent analysis if required. Data acquired was
stored on 3.5 in floppy disks and data analysis performed on
an integral Hewlett Packard personal computer (model #65).
Chromatographic separations were achieved on a wide pore
silica based butyl reverse phase columns purchased from
Synchrom. These columns have a bead size of 7 microns and a
pore diameters of 300 A which allow for the for the separation
of proteins to a maximum Mr of 300 kDa. Two sizes of this
type column were employed, a 4.1 X 250 mm column or a
narrowbore column of 2.1 X 250 mm I.D. The conventional
column (4.1 X 250 mm) was used for those experiments where
larger amounts of protein, usually >400 pmol,were needed.
Such was the case in those experiments where the identity of
the proteins purified by RP-HPLC was determined by
coelectrophoresis of eluted peaks on 2D-PAGE. The narrowbore
column was primarily used when <400 pmol of total protein was
needed as was the case for microsequencing of purified
proteins. The narrowbore column operated at approximately one
third the flow rate and at least four times the sensitivity of
the conventional column (Simpson and Nice, 1987), which
allowed for the analysis of 100 pmol of ribosomal protein.
Both columns used the same bonded phase butyl packing material
and nearly identical separations resulted.
Mitochondrial ribosomal proteins were resolved on the
silica-C4 HPLC column by a gradient elution of increasing
proportions of 2-propanol versus water over a 10 h time

119
period. Both the aqueous and organic solvents contain 0.1%
TFA, as a counter ion to charged amino groups. Flow rates
were 250 nl/min from 0 to 260 min and then reduced to 200
jil/min until the end of the analysis for the conventional
column. For the narrowbore RP-HPLC column, a flow rate of 200
nl/min was maintained for the first 100 min while the samples
were loaded onto the column in multiple injections at 1%
organic solvent. During gradient elution the flow rates were
80 nl/min from 100 to 260 min and then reduced to 64 nl/min
until the end of the analysis. The gradient profile for both
columns was as follows: 0 to 100 min, 1% organic; to 120 min,
12%; to 140 min, 19%; to 155 min, 22%; to 260 min, 28%; to 570
min, 42%; to 600 min, 55%; to 630 min, 75%; and to 635 min,
25% 2-propanol. The column was maintained at a constant
temperature of 35°C during an analysis. Samples were injected
at 1% 2-propanol by multiple injections until all the samples
had been injected. This was possible because proteins exhibit
high binding capacities below critical organic solvent
composition on reverse phase supports. Therefore it was
possible to concentrate large sample volumes directly on the
column before recovery of proteins by gradient elution (Nice
et al., 1979). Samples were collected into 1.5 ml plastic
screw cap tubes at 1 to 5 min intervals with the aid of a
fraction collector.

120
Gas Phase Protein Sequence Analysis:
Determination of primary sequence of ribosomal proteins
was done by Edman (1956) degradation. Edman degradation is a
repetitive reaction in which the alpha amino group of a
polypeptide chain is chemically coupled to phenyl isothio¬
cyanate (PITC) under alkaline conditions with trimethyl amine
(TMA). The amino terminal derivative is then cleaved in the
presence of trifluoroacetic acid (TFA). resulting in an
ani1inothiazolinone (ATZ) derivative. This derivative is then
converted to a stable phenylthiohydantoin (PTH) amino acid in
the presence of TFA and water, which was then analyzed by RP-
HPLC.
Polypeptide sequence analysis was performed with a 470
gas phase protein sequenator with a 120A on line PTH-HPLC
analyzer (Applied Biosystems). Samples sequenced were bound
to polyvinylidene difluride (PVDF) membrane by electroblotting
proteins from 1D-SDS gels.
Proteins or peptides were sequenced and the resulting
PTH-amino acids were analyzed on a model 120A PTH-HPLC
analyzer using programs 03RPTH. This chromatographic program
consisted of a 2.1 X 220 mm C18, HPLC column (model PTH C18,
ABI, Foster City, CA). The bead size of the C18 column
packing was 5 microns. The aqueous solvent was 60 mM sodium
acetate, pH 4, and the organic solvent was acetonitrile with
3.5% tetrahydrofuran. The flow rate was 200 nl/min and
separation of PTH residues was achieved by an increasing

121
gradient of organic solvent. The gradient profile for this
HPLC method was as follows: 0 to 9 min, 8%; to 11 min, 15%;
to 11.3 min, 16%; to 29 min, 36%; to 40 min, 36%; to 40.1 min,
80%; and continued at 80% until the end of the chromatogram at
47 min. Repetitive yields were determined by comparison of
peak heights of Val, Leu, Glu, to peak heights of these same
residues as they occur later in the sequence. The decrease in
yield of these residues versus the number of cycles
approximated the repetitive yield.
CNBR Digestion of Ribosomal Protein
To obtain internal amino acid sequence of a ribosomal
protein the protein was chemically digested with CNBR. In 70%
formic acid CNBR cleaves polypeptides on the carboxyl side of
methionyl residues (Gross and Witkop, 1961). The CNBR
peptides were resolved on a 15% polyacrylamide Laemmli gel
then el ectrobl otted onto PVDF membrane for gas phase
microsequencing. This method was applied to one ribosomal
protein, S5, for which amino-terminal sequence could not be
obtained, presumably due to chemical blockage of the amino
terminus. The protein was first isolated in pure form by
narrow bore RP-HPLC and the fractions containing the pure
protein pooled and concentrated to near dryness (approximately
a 10 nl volume) in a speed vac centrifuge. The sample was
brought to a 20 ^1 volume with water and then 88% formic acid
(ACS grade, Fisher Scientific) was added to a final
concentration of 70% formic acid with a resulting volume of

122
100 jil. Approximately 1 mg of CNBR powder was quickly added
to the sample under a fume hood. After the CNBR was in
solution a stream of N2 gas was passed over the sample for 15
sec and the tube was capped quickly. The sample was wrapped
in foil to exclude light and allowed to digest at room
temperature for 16 h.
To resolve the CNBR peptides the sample was prepared for
gel electrophoresis. Laemmli sample buffer (30 *il), with no
bromophenol blue dye, was added to the sample and concentrated
to near dryness in a speed vac centrifuge. Once the volume
was approximately 30 jil, HPLC grade water was added to a final
volume of 100 nl and speed vac concentrated again to near
dryness. This step was repeated once more to reduce the
concentration of the formic acid. A small volume (10 |il) of
Laemmli sample buffer, with dye added, was again added to the
sample and 5% TEA (triethanolamine) was added to the sample
(approximately 60 *il) until the pH indicator dye turned blue
to indicate the sample was slightly basic and then heated at
100°C for 4 min prior to electrophoresis. The CNBR peptides
were resolved on a 15% polyacrylamide Laemmli gel and then
electroblotted onto PVDF membrane as described below for amino
acid sequence analysis.
Electroblottina of Ribosomal Proteins onto PVDF
Ribosomal proteins or CNBR peptides of ribosomal proteins
were resolved on one dimensional SDS Laemmli gels and
electroblotted onto PVDF membrane for amino acid sequence

123
analysis. Following electrophoresis, a wetted PVDF membrane
was placed, in close contact, on one side of the 1D-SDS gel
and then sandwiched between two sheets of wetted Whatman
paper. The membrane was first wetted briefly in 100%
methanol, then blotting buffer (0.025 M Tris-HCl, pH 8.3 , 20%
HPLC grade methanol, 0.193 M glycine, and 0.01% SDS) for 10
min. Blotting was done in blotting buffer in an
electroblotting apparatus (Bio-Rad, trans-blot model) at 60 V
for 24 h at 4°C with the gel oriented so that the PVDF
membrane was closest to the cathode. The electroblotted PVDF
membrane was then lightly stained with Coommasie blue (0.1% in
50% methanol) stained for 5 min and destained in destain
solution (50% methanol, and 2% acetic acid, both HPLC grade)
for approximately 10 min until polypeptide bands were visible.
Bands of interest were excised with a sterile scalpel from the
membrane and placed in sterile vials and purged with nitrogen
gas and stored at -20°C until analyzed.
Analysis of One and Two-Dimensional Immunoblots of 28S
Ribosomal Proteins
Small subunit ribosomal proteins were resolved on either
a 1-dimensional Laemmli gel (12% polyacrylamide) or 2-
dimensional urea gel as described above. Small subunit
proteins were electrophoresed on the Laemmli gel in a wide
lane (5 cm) forming a curtain of resolved protein. Both types
of gels were then electroblotted onto sheets of nitrocellulose
or PVDF membrane. Electroblotting for either support was

124
identical as described above with the exception that the
nitrocellulose need not be prewetted in 100% methanol.
Following transfer onto the support, strips were cut (5 X 200
mm) parallel to the direction of electrophoresis to obtain
resolved 28S ribosomal proteins for immunoblot analysis.
Electroblotting 2-dimensional gels did require
equilibration of the gel in copious quantities of blotting
buffer for 2 h before transfer could begin to remove excessive
amounts of urea. Electroblotting of 2-dimensional gels was
performed in the same manner as for Laemmli gels.
Immunoblot analysis for proteins electroblotted onto
nitrocellulose or PVDF supports were done in an identical
manner. Blots were first incubated for 30 min at 4°C in
blocking solution (3% Bovine serum, 150 mM NaCl, 10 mM Tris-
HC1, pH 7.5, and 0.01% sodium azide). Rabbit serum containing
monospecific antibody against S5 was diluted 1:100 in
incubation buffer (same composition as blocking solution with
0.5% Triton X-100, .01% SDS added) for analysis of 1D-
Immunoblot strips. Anti S5 rabbit serum was depleted of
antibodies against bovine liver proteins and E. coli lysates,
as described below, and also diluted to a final ratio of 1:100
with incubation buffer prior to 2-dimensional immunoblot
analysis.
Individual blots were incubated with the appropriate
diluted anti S5 serum overnight at 4°C with gentle shaking.
28S Ribosomal strips and 2-dimensional blots were incubated in

125
approximately 1.5 and 15 ml of diluted serum, respectively.
Blots were then washed for 20 min, 3 times in cold incubation
buffer. A second anti-rabbit IgG (Fc) antibody alkaline
phosphatase conjugate, was diluted 1:7500 with incubation
buffer and incubated with the blots for 1 h at room
temperature with gentle shaking. Blots were washed twice at
room temperature while shaking with TBSA (150 mM NaCl, 10 mM
Tris-HCl, pH 7.5, and 0.01% sodium azide) and a final wash
with TBS (same as TBSA but no sodium azide present).
Immunoreactive proteins were visualized using a color reaction
provided in a ProtoBlot Immunoscreening Kit (Cat# P3710,
Promega, Madison WI). Alkaline phosphatase conjugated to the
Fc portion of a anti-rabbit IgG catalyzed the
dephosphorylation of the substrate, BCIP (5-bromo-4-chloro-3-
indolyl phosphate), which in turn reacted with NBT (nitro blue
tetrazolium) to form a purple color, indicating
immunoreactivity by the primary antibody.
Depletion of Anti S5 Rabbit Serum of Bovine Liver and E. coli
Lvsate Cross Reacting Antibodies
Rabbit serum containing monospecific antibodies against
mitochondrial ribosomal protein S5, were depleted of
antibodies that would bind to epitopes in both Bovine liver
and E. col i lysates. This was done to reduce background
signals for 2-dimensional immunoblot analysis with this rabbit
serum. This was accomplished by passage of the rabbit serum,
diluted 1:10 in incubation buffer, through 2 columns each

126
containing either liver or E. coli lysates conjugated to
Reacto-Gel (HW# 65F, Sigma).
Conjugation of the Bovine liver lysate was accomplished
by first chopping 8 g of commercially purchased frozen beef
liver in 20ml of ice cold 0.1 M sodium borate buffer, pH 8.5
with 2% SDS. The liver was finely homogenized for a few
seconds on ice with a small Tekmar tissue homogenizer. The
volume was adjusted to 40 ml with 0.1 M sodium borate buffer
with 2% SDS. The liver homogenate was then heated to 100°C
for 5 min and placed in a 50 ml conical tube and centrifuged
for 10 min at 1800 rpm in a I EC table top centrifuge at room
temperature. 20ml of the supernatant was removed, added to 15
ml of React-Gel and adjusted to a final volume of 50 ml with
cold 0.1 M sodium borate buffer. The coupling reaction was
allowed to proceed for 60 h while stirring at 4°C. A small
tubular glass column (1.5 X 8 cm) was packed with the liver
lysate coupled gel, washed 5 volumes of 1 M NaCl, then with an
equal volume of TBS. The column wash was repeated once more
and the column was sealed and stored in TBS at 15°C until
used.
The preparation of the E. col i lysate column was
identical to that described for the liver lysate column. Only
the preparation of the lysate differed. One liter of E. coli
Y1090 was grown overnight at 37°C in Luria Broth with 0.2%
maltose, 10 mM MgS04, 10 jig/ml tetracycline, and 40 ng/ml
ampicillin. The cells were collected by centrifugation at 800

127
rpm for 5 min and solubilized gently in 20ml of TBS at pH 8.0.
Lysozyme (Sigma) was added to concentration of 5 mg/ml and
incubated for 1.5 h at room temperature. Tween 20 was added
to a final concentration of 1% and vortexed repeatedly. 20%
SDS was added to a final concentration of 2% and mixed with a
vortex mixer again and allowed to incubate overnight at 4°C
without shaking. The mixture was placed in a 100°C water bath
for 5 min, then centrifuged at 1800 rpm for 10 min to
sediment cellular debris. An 8 ml aliquot of the supernatant
was diluted to 35 ml with 0.1 M sodium borate buffer and added
to 15 ml of the Reacto-Gel and the coupling reaction done as
described above.
The rabbit serum containing the monospecific anti S5
antibody was diluted 1:10 with cold TBS. The diluted serum
was first passed through the E. coli lysate column which was
equilibrated with 2 volumes of TBS. All column manipulations
were done at 15° C in a cold box and all solutions used were
also kept at that temperature. The column eluant passed
through a 5 mm flow cell and the detector (ISC0, model#UA-5)
signal was monitored at 254 nm and recorded on a Fisher
Recordal 1 (model# 5000) strip chart recorder. Following
equilibration, a baseline recording was achieved, the diluted
serum was added to the open column and passed through the
column by gravity alone. When the diluted serum passed into
the column bed completely the column reservoir was filled with
TBS. The antibody peak was collected until the signal

128
returned to the baseline value. The column was regenerated by
passing 20ml column elution buffer (0.5 M HPLC acetic acid,
and 1% Dioxane) followed by an equal volume of TBS through the
column. A peak of bound antibody, approximately one tenth the
volume of the free antibody peak, eluted with the elution
buffer. The column was then quickly neutralized with 1 volume
(20ml) of 2 M Tris and the pH of the eluant was checked until
it was above pH 9. Two volumes of TBSA were then passed
through the column and the column stored three fourths full of
TBSA at 15°C for future use.
The free antibody peak collected was next passed through
the Bovine liver lysate column in the same manner as described
above. Following passage through both columns the free
antibody peak was diluted to a final ratio of 1:100 with
incubation buffer and E. coli lysate was added to the serum to
a final concentration of 10% (v/v).
Results
RP-HPLC of 28S Ribosomal Proteins and their Identification bv
2D-PAGE Analysis
Photoaffinity methods identified S5 as the mitochondrial
ribosomal protein responsible for specifically binding GTP.
Reverse phase HPLC methods were employed to isolate and purify
this protein and other 28S ribosomal proteins. The first task
was to identify which peak S5 corresponded to and to assess
its purity. The second task was to isolate adequate amounts

129
of pure S5 for gas phase chemical microsequencing to obtain
partial amino acid sequence information for this protein.
Small subunit ribosomes were extracted in 66% acetic acid
as described in Methods to remove rRNA and injected onto a
wide pore (300 A) butyl (C4) reverse phase HPLC column (4.1 X
250 mm). The extracted ribosomes were loaded onto the column
in a series of 12, 100 nl injections spaced 5 min apart until
approximately 1600 pmol of ribosomal protein was applied to
the column at 1% 2-propanol. The ribosomal proteins were
eluted from the RP-HPLC column with a gradient of increasing
concentration of 2-propanol as described in Methods. Eluted
fractions were collected at 2.5 min intervals and 10% of each
fraction was removed for ID-PAGE analysis, and the remainder
left uncapped overnight in a fume hood to allow for the
evaporation of the 2-propanol from the samples. Thirty nl of
50% ultra pure glycerol was added to the samples and then
evaporated in a Savant speed vac apparatus to approximately
one fourth their original volume or about 200 to 150 jil. The
samples were overlaid with a stream of N2 gas and quickly
capped and stored at 15°C in the dark until needed for 2D-PAGE
analysis.
Reverse phase high pressure liquid chromatographic
separation of mitochondrial 28S ribosomal proteins has been
developed to the extent that the majority of the ribosomal
proteins can now be resolved to purity (figure 5-1). Co¬
electrophoresis of eluted peaks from the RP-HPLC C4 column

130
15
Figure 5-1 Reverse phase high pressure liquid chromatogram of
1.6 nmol of acetic acid extracted 28S ribosomes resolved on a
wide pore (300 A) silica bonded butyl column. The ribosomal
proteins were injected into the chromatograph in several small
iniection volumes (100 ni X 20) every 5 min at 1% 2-propanol
(this part of the chromatograph is not shown). The proteins
were then resolved with a increasing gradient of 2-propanol
(%0rg) versus water both with 0.1% TFA as described in
Methods. Peaks were detected at 214 and 280 nm simultaneously
and the 280 nm signal (at 0.1 AUFS) is shown in this figure.
The eluted peaks are numbered sequentially as they elute from
the column. The proteins comprising these peaks were
identified by coelectrophoresis on 2D-PAGE gels with 28S
ribosomes in the following figures (figure 5-2 A to Q). The
peak numbering system used in this figure was used in this
identification process. Fractions were collected at 2.5 min
intervals starting at 110 min and continuing until 500 min or
the end of the chromatogram. In table 5-1 the fractions and
peaks selected for 2D-PAtlE identification and their identities
are summarized.

131
with 28S ribosomal proteins on 2D-PAGE gels has allowed for
the identification of the proteins in 27 out of 31 peaks
(table 5-1). S5 was a late eluting peak (#29) that contained
no other proteins. The identity was determined by 2D-PAGE co¬
electrophoresis (figure 5-2 A to Q, and table 5-1) and
immunoblot analysis (figure 5-5) with an anti S5 antibody that
was confirmed to predominately recognize S5 by 2D-PAGE
immunoblot analysis (figure 5-4).
Amino-terminal sequence has been obtained for four small
subunit ribosomal proteins (table 5-2) isolated by RP-HPLC.
Sequences were obtained in all cases with the use of PVDF
membrane as the solid support for micro gas-phase sequence
analysis. The technique for application of the samples to the
support membrane varied in an effort to find a method that
would yield optimal results (table 5-1). For those proteins
eluted as pure peaks the samples could be either spotted
directly onto the PVDF disks or allowed to bath in the peak
eluant while shaking overnight. Both methods provided
satisfactory results but samples must be allowed to stay in
contact with the membrane for several hours to allow for
complete absorption of the sample onto the membrane prior to
sequencing. Another precaution that was necessary to prevent
chemical blockage of the amino terminus by TFA present in the
HPLC mobile phase was to not allow the proteins to remain in
TFA for more than 48 h.

132
S5 was the only small subunit protein sequenced so far
that apparently was naturally blocked at its amino-terminus
preventing N-terminal sequence analysis. Amino-terminal
sequencing of S5 was attempted twice and both times no
sequence was obtained, even though analysis of the sample
remaining on the PVDF membrane revealed at least 17 pmol
(table 5-1) of protein as determined by acid hydrolysis
followed by amino acid analysis on a Beckman 6300 AA analyzer
(data not shown). The chemical nature of the amino-terminal
blockage is unknown.
Identification of S5 and other ribosomal proteins eluted
from the RP-HPLC column was accomplished by coelectrophoresis
of eluted peaks with 28S ribosomal proteins on 2D-
polyacrylamide gels. A 10 to 20 molar excess of HPLC resolved
ribosomal proteins (figure 5-2) were added to a minimal amount
of acetic acid extracted 28S ribosomes (30 pmol) and were
dialyzed versus 2D-PAGE sample buffer overnight as described
in Methods. Following 2D-PAGE resolution and silver staining
of the gels, identification of the ribosomal proteins eluted
by HPLC was made by comparison of the enhanced staining of
those ribosomal proteins, present in 10 to 20 molar excess, to
the background amounts of 28S ribosomal proteins.
Additionally, a small amount (approximately 160 pmol) of the
RP-HPLC resolved proteins were electrophoresed in a lane
parallel to the second dimension to aid in identifying
enhanced peaks. In figure 5-2 (A to Q) are 2D-PAGE gels with

133
the identity of the ribosomal protein present in the
corresponding RP-HPLC peak. Table 5-1 summarizes the identity
and purity of the proteins eluted from the RP-HPLC column. S5
was identified (figure 5-2 J) as peak #29, eluting as a pure
peak at 36% 2-propanol or 428 min (figure 5-1).
Identification of $5 RP-HPLC Peak bv Western Blot Analysis
Usina a Monospecific Antibody Raised Against $5
A monospecific antibody was raised against S5 and other
small and large mitochondrial ribosomal proteins by
Pietromonaco et al. (1986). Monospecific antibodies were
prepared using proteins extracted from 2-dimensional gels
bythe acetic acid method of Bernabeau et al. (1980). A rabbit
was immunized with S5 extracted from gel plugs cut from five
2-dimensional gels. Each 2-dimensional gel was prepared
with approximately 270 pmol of 28S ribosomal protein. This
rabbit required 4 booster injections of S5 protein from the
same number of gel plugs except, for the last injection where
10 gel plugs were used, before antibody was detected in the
serum. The total period of time required for immunization of
S5 in this rabbit was 6 months. Pietromonaco et al. (1986)
reported a strong immunoreaction of a ribosomal protein on a
1-dimensional immunoblot corresponding to the Mr of S5.
Serum collected from this rabbit was stored at -20°C for
approximately 5 years before use in this study. To ensure the
quality of this sera immunoreactivity of 1-dimensional blots
of 28S ribosomal proteins were examined. Frozen 350 ^1

of ribosomes was used
coelectrophoresis. The
column and identified
chromatographic run shown
Figure 5-2 Two-dimensional PAGE analysis of 28S ribosomal
proteins eluted from a RP-HPLC butyl column, coelectrophoresed
with a minimal amount of acetic acid extracted 28S ribosomes.
In gels B to J, 38 pmol of acetic acid extracted ribosomes
were coelectrophoresed with approximately 5 to 20 molar excess
of ribosomal proteins eluted from the butyl RP-HPLC column
shown in figure 5-1 on a 1.5 X 160 X 200 mm slab gel. In gels
L to Q 23 pmol of extracted ribosomes were used and
coelectrophoresed with ribosomal proteins eluted from the
butyl column as in the preceding gels. These 2D-PAGE gels
were smaller in size, 1.5 X 80 X 100 mm. The same preparation
for both RP-HPLC and 2D-PAGE
proteins eluted from the RP-HPLC
by 2D-PAGE were from a single
in figure 5-1. In every 2D-PAGE gel
a portion (approximately 5% v/vj of the eluted peak was run in
a lane on the second dimension slab gel, parallel to the
second dimension, to show the migration of the eluted
protein(s) in the urea/SDS slab gel. Proteins isolated by RP-
HPLC that were identified as an enhanced protein spot
migrating approximately the same distance on the second
dimension as the one dimensional lanes. These are identified
by protein name on each figure. These fractions are labeled as
to their peak (P, figure 5-1) and fraction (f) number. In
table 5-1 the retention times and protein identities
corresponding to these fractions from the RP-HPLC (figure 5-1)
are summarized. Gels A and K are electrophoretic patterns of
28S ribosomal proteins, without any HPLC fraction added,
electrophoresed under the same electrophoretic conditions for
gel B to J and L to Q respectively.

135
A
5 L,3
' $
I I-
ir
16*
>10
o'2
O
21.
23c
14
e>l 8°' 7
022
25 26
o o
270
28oQ CP30 032
31
O
33
- 94
- 67
- 43
- 30
- 20.1
- 14.4
P 7
f 31 M B
— I I • - —•
S 22
Figure 5-2 A & B Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction
(f)#31 of peak (p) #7 (panel B) as shown in figure 5-1. In
panel A the electrophoretic pattern for the two-dimensional
separation of 28S ribosomes without any added HPLC fractions
is shown to aid in the identification of ribosomal proteins
for gels B to J. The same molecular weight markers (M) used in
figure 3-2 were used in the following figures (A to Q).

136
P 8 P12 C
f33 M f44
PI5 D
f 58 M
I I
Figure 5-2 C & D Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction #33
of peak #8 and fraction #44 of peak #12 (panel C) as shown in
figure 5-1. In panel D, RP-HPLC fraction #58 of peak #15 was
coelectrophoresed with 28S proteins. Total protein (TP) and
molecular weight markers (M) are also shown.

137
PI6 E
f 59
/
- 94
- 67
- 14.4
P14 P2I
f 56,73,74
I / /
PI4 F
f 55
/
Figure 5-2 E & F Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction #19
of peak #16 (panel E) shown in figure 5-1. In panel F, RP
HPLC fractions #55 and 56 of peak #14 and fraction #73 and 74
of peak #21 were coelectrophoresed with 28S proteins.

138
P9
f36
I
•
ll Mfci ^
â– 
• -
— S29— A
P20.25.3I
P20
f 70,88,149
69 M
/ / /
1 /
—SI —
P*
1+ S6 —
*
- S7
•
* •
m _ oo*z
o cO
S23 P*
G
-94
-67
-43
-30
-20.1
-14.4
H
Figure 5-2 G & H Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction #36
of peak #9 (panel G) shown in figure 5-1. In panel H, RP-HPLC
fractions #b9 and 70 of peak #20, fraction #88 of peak #25,
and fraction #149 of peak #31 were coel ectrophoresed with 28S
proteins.

139
P22.28
f 78,122/3
/ //
P23
f 82 M
\ /
I
- 94
- 67
- 43
- 30
- 20.1
- 14.4
P13b P29
f47 f 130
\ \
J
Figure 5-2 I & J Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction #78
of peak 22, fractions #122, and 123 of peak #28, and fraction
#82 of peak #23 (panel I) as shown in figure 5-1. In panel J,
RP-HPLC fraction #47 of peak #136, and fraction #130 of peak
#29 were coel ectrophoresed with 28S proteins.

140
K
8
4
O 5
O
°7S>!3
O o> O
'
I 3
8
5
8
Ol
§022
2
Z\70 025
2J«
26
28~ 3 |O¿30 0 32
O
33
— 9 4
67
-43
- 30
- 20.1
- 14.1
L
Figure 5-2 K & L Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fractions
(f) #66, and 67 of peak (p) #19 and fractions #16 and 17 of
peak #3 (panel L) as shown in figure 5-1. In panel K the
electrophoretic pattern for the two-dimensional separation of
28S ribosomes without any added HPLC fractions is shown to aid
in the identification of ribosomal proteins for gels L to Q.

141
M
p4 P27
-94
- 67
-43
- 30
- 20.1
- 14.4
Figure 5-2 M & N Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction #21
of peak 4, and fractions #100, and 101 of peak 27 (panel M) as
shown in figure 5-1. In panel N, RP-HPLC fractions #38 and 39
of peak #10 were coelectrophoresed with 28S proteins.

142
pío O
P
Figure 5-2 0 & P Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fractions
#40 and 41 of peak #11, and fraction #86 of peak #9 (panel 0)
as shown in figure 5-1. In panel P, RP-HPLC fraction #89 of
peak #25 and fractions #55 and 56 of peak #14 were
coelectrophoresed with 28S proteins.

143
Figure 5-2 Q Two dimensional PAGE electrophoresis of 28S
ribosomal proteins coelectrophoresed with RP-HPLC fraction #69
and 70 of peak #20, and fractions #138 and 139 of peak #30
(panel Q) as shown in figure 5-1.

144
Table 5-1
Summary of Peak Identification for RP-HPLC of 28$ Ribosomal
Proteins
Peak Fraction Retention Protein Pure 2D-PAGE
Number3
Number3
T i me3
(min) Identitvb (Y/N)b
Gelb
3
16,
17
147.5
-
152.5
S26 Y
L
4
21
160.0
-
162.5
S32
Y
M
7
31
185.0
-
187.5
S22
Y
B
8
33
190.0
-
192.5
S30
Y
C
9
36
197.5
-
200.0
S29
Y
G, P
10
38,
39
202.5
-
207.5
S10
N
0
11
40,
41
207.5
-
212.5
S10
N
P
12
44
217.5
-
220.0
S18
Y
C
13b
47
220.0
-
222.5
S12, S14
N
J
14
55,
56
245.0
-
250.0
S31
Y
F, N
15
58
252.5
255.0
S7, S12
S15, S17
S29
N
D
16
59
255.0
257.5
S7, S12
S15, S17
N
E
19
66,
67
272.5
-
277.5
S7
Y
L
20
69,
70
280.0
-
285.0
S23
Y
H, Q
21
73,
74
290.0
—
295.0
S23 major
S6 minor
N
F
22
78,
79
302.5
-
305.0
S25
Y
I, R
23
82
312.5
315.0
S21 major
S28 minor
N
I
25
88,
89
327.5
332.5
56 major
57 minor
N
H, N
26
91,
92
335.0
-
340.0
S16 major
N
R
27
100,
101
357.5
-
362.5
S8
Y
M
28
122,
123
412.5
-
417.5
HMr Protc
N
I
29
130
432.5
-
435.0
S5
Y
J
30
138,
139
452.5
-
457.5
S4
Y
Q
31
149
480.0
482.5
SI
Y
H
!Refer to figure 5-1.
bRefer to figure 5-2.
cHMr Prot. = High molecular weight (< 70 kDa) protein that are
not ribosomal proteins.

145
aliquots of nonimmune and anti S5 rabbit sera were diluted
1:100 with cold incubation buffer and incubated with strips of
electroblotted 28S ribosomal proteins resolved on a Laemmli
gel as described in Methods. In figure 5-3, a wide protein
band from 37.5 to 41.5 kDa, corresponding to the Mr of S5 and
possibly another protein, was immunoprecipitated by the anti
S5 sera. Other minor bands were immunopre-cipitated but were
no different than those seen in the nonimmune sera (figure 5-
3).
To determine if the anti S5 serum recognizes S5 or
possibly other 28S proteins, a 2-dimensional blot treated with
S5 antiserum that had been passed through Bovine liver and E.
coli lysate columns was examined. In figure 5-4, S5 was
clearly the dominant protein recognized by the anti S5 serum,
but to a lesser extent S6 was also recognized. Other minor
proteins recognized were S15 and to a lesser extent S8, with
only trace amounts of S2 and S3 being recognized. This
suggests that the wide protein band noted above in the 1-
dimensional immunoblot corresponds to $5 and S6. All the
other bands which were also seen in the nonimmune serum
correspond to other ribosomal proteins. All the minor bands
that were detected in approximate equal amounts using either
the nonimmune or the immune sera by 1-dimensional immunoblot,
correspond well in Mr to S2, S3, S4, S8, S12, S15, and S18,
which appear as minor spots on the 2-dimensional immunoblot.
This observation suggests that this rabbit serum, raised

146
NI Imm S
—
-94
“67
54 “• -~
55 — |
MU
-43
—
-30
-20.1
-14.4
Figure 5-3 Immunoblot of 28S bovine ribosomal proteins with
an anti S5 monospecific rabbit sera. 28S Ribosomes were
electrophoresed on a 12% SDS-PAGE and electroblotted onto PVDF
membrane. Strips of the blotted membrane containing
approximately 25 pmol of ribosomal protein were incubated with
non-immune (NI) and immune (Imm) rabbit sera, diluted 1:100
with low salt incubation buffer (150 mM NaCl) with 3 % bovine
serum albumin at 4°C. Total 28S proteins, fast green stained
(S), are shown on the right with molecular weight markers.
Due to shrinkage of the PVDF membrane during fast green
staining, immunoblotted and the corresponding stained proteins
were marked for identification purposes. Binding of antibody
to protein was visualized by a color reaction with the use of
a second anti-rabbit IgG conjugated to alkaline phosphatase as
described in Methods. Molecular weight markers used were the
same ones described in figure 3-2. For additional
experimental details see Methods.

147
94
43
20
Figure 5-4 Two-dimensional immunoblot analysis of 28S bovine
ribosomal proteins with an anti S5 monospecific rabbit sera.
28S Ribosomes were electrophoresed on a two dimensional PAGE
and electroblotted onto PVDF membrane. Blotted membranes
containing approximately 230 pmol of ribosomal protein were
incubated with immune rabbit sera depleted of antibodies that
bind liver and E. col i lysates. The depleted serum was
diluted to a final concentration of 1:100 with low salt
incubation buffer (150 mM NaCl + 3% bovine serum albumin) and
incubated with the blotted membrane at 4°C overnight. Binding
of antibody to protein was visualized by a color reaction with
the use of a second anti-rabbit IgG conjugated to alkaline
phosphatase as described in Methods. Molecular weight markers
used were the same ones described in figure 3-2. S5 was the
protein primarily recognized with this sera though S6 and S15
were also recognized, to a lesser degree. For additional
experimental details see Methods.

148
against S5, will primarily recognize S5 but it is expected
that other 28S ribosomal proteins, especially S6, will also be
recognized to a lesser extent.
Immunoreactivitv of 28S Ribosomal Proteins Resolved bv RP-HPLC
Acetic acid extracted 28S ribosomes were resolved by RP-
HPLC, and eluted fractions were western blotted onto PVDF
membrane and probed with the anti S5 serum to verify the
identity of the S5 peak. Small subunit ribosomes (658 pmol)
were acetic acid extracted and resolved on a narrow bore (2.1
X 250 mm) C4 column as described in Methods. The column
eluant was collected in 5 min fractions and 2.5% of each
fraction was pipetted onto prewetted PVDF membrane in a 96
well dot blot apparatus. The eluant was allowed to incubate
on the PVDF membrane in the dot blot apparatus for 2 h at 15°C
with gentle shaking. The eluant was evacuated from the
membrane under gentle vacuum for about 5 sec and allowed to
dry in air for 10 min. The blot was incubated with blocking
solution (3% Bovine serum) for 30 min at room temperature
while shaking, and then anti-S5 rabbit serum diluted 1:100
with incubation buffer was added and shaken gently at 15° C
for 10 h. Immunoreactive proteins were visualized by means of
the alkaline phosphatase conjugated anti-rabbit antibody as
described in Methods. Five percent of each eluted RP-HPLC
fraction was also electrophoresed on a 12% polyacrylamide
Laemmli gel to determine the purity of the fraction. In
figure 5-5, dot immunoblots show a strong immunoreactivity of

149
the late eluting chromatographic peak centered at 425 min.
This verifies the identity of this chromatographic peak as
containing S5. This peak was pure (figure 5-5, gel panel) and
suitable for chemical sequence analysis. Weaker signals were
seen in fractions containing S6, S8, S15, and S18, which were
expected, due to the results seen in 1 and 2-dimensional
immunoblots (figures 5-3 and 5-4). S5 electrophoresed as a
wide band containing two closely resolved bands centered at 38
kDa.
To ensure that both bands are forms of S5, fractions from
415 to 440 min inclusive were pooled and 5% of this pooled
material was analyzed by 1-dimensional immunoblot analysis
(figure 5-6). This aliquot contained approximately 33 pmol of
antigen and was electrophoresed on a 12% polyacrylamide
Laemmli gel, then electroblotted onto PVDF membrane.
Immunoblot analysis with anti-S5 serum diluted 1:100 with
incubation buffer was done as described above and both closely
resolved bands were strongly and equally immunoreacti ve
(figure 5-6). This strong and equal recognition of both bands
suggests strongly that both are forms of S5.
Amino Acid Sequence Analysis of $5 and Other 28S Ribosomal
Proteins
Amino-terminal amino acid gas phase microsequencing of
proteins was undertaken for a number of mitochondrial small
subunit ribosomal proteins, including S5. All ribosomal

Figure 5-5 Immunoblot analysis of 28S bovine ribosomal
proteins separated by RP-HPLC with anti S5 monospecific
rabbit sera. Acetic acid extracted 28S ribosomes (658 pmol)
were separated on a wide pore silica based butyl HPLC column
(2.1 X 250 mm) as described in Methods and shown previously in
figure 5-1. The eluant from the column was collected between
160 to 560 min at 5 min intervals. Approximately 1% of the
eluant (6.6 pmol) from each fraction was blotted onto PVDF
membrane with the use of a 96 well dot blot apparatus. The
blotted membrane was incubated with anti-S5 monospecific
rabbit sera, diluted 1:100 with low salt incubation buffer
(150 mM NaCl) containing 3 % bovine serum albumin at 4°C
overnight. Binding of antibody to protein was visualized by
a color reaction with the use of a second anti-rabbit IgG
conjugated to alkaline phosphatase as described in Methods.
Approximately 10% of each fraction from the HPLC eluant was
analyzed by 12% SDS-PAGE as shown at the bottom of the figure.
The identity of many of the proteins is also indicated, when
known. Molecular weight markers used are the same ones
described in figure 3-2.

.01 AU 280nm
Time Cmin]

152
H
>
Z
94-
6 7-
43-
30-
2 0-
S 5
03
r
O
-94
-67
-43
-30
-20
Figure 5-6 Immunoblot analysis of S5 protein isolated by RP-
HPLC. Fractions eluted from 415 to 440 min inclusive, were
pooled and approximately 5% was electrophoresed by 12% SDS-
PAGE and electroblotted onto PVDF as described in Methods.
The blotted membrane was incubated with anti-S5 monospecific
rabbit sera, diluted 1:100 with low salt incubation buffer
(150 mM NaCl) with 3 % bovine serum albumin at 4°C overnight.
Binding of antibody to protein was visualized by a color
reaction with the use of a second anti-rabbit IgG conjugated
to alkaline phosphatase as described in Methods. The
immunoblotted protein (BLOT) is compared to a silver stained
(STAIN) lane to indicate the purity of the protein fraction.
The chromatographic peak contained greater than 98% pure S5
protein which was recognized by the anti-S5 monospecific
antibody. The remainder of this pooled sample was used to
obtain amino acid sequence by Edman degradation chemistry.

153
proteins were sequenced on PVDF membrane supports and table 5-
2 summarizes the methods employed. Proteins isolated in pure
form from the RP-HPLC were deposited onto discs (1 cm
diameter) of PVDF membrane by either being spotted onto or
bathing discs in the eluant. If pure fractions were not
available, the protein or peptides were electrophoresed and
electroblotted onto PVDF for sequencing as described in
Methods.
For one protein, S5, amino terminal sequencing failed to
produce any sequence even though two attempts were made under
conditions where other proteins were successfully sequenced
(Table 5-2). Chemical cleavage was then done to obtain
internal sequence information and was accomplished by chemical
digestion at methionyl residues with cyanogen bromide. This
generated 6 peptides (figure 5-7), in addition to the full
length S5 protein. All six CNBR peptides were sequenced
successfully. Several were redundant sequences, suggesting
partial cleavages with the chemical agent, but three unique
sequences were found (table 5-3). One disadvantage to
sequencing proteins blotted onto PVDF supports is a reported
loss of tryptophane residues during electrophoresis or
blotting (Matsudaira, 1987). Sequence analysis of the 13 kDa
CNBR peptide of S5 revealed that at residue 5 no appreciable
amount of PTH-AA could be detected above background levels
(figure 5-8 A). There was apparently PTH-Trp, but the amount
was not significantly above background levels. Also a new

154
MWM-
â–  S 5 CNBR PEPTIDES
94 —
6 7 —
4 3 —
kl
3 : — •
20.1— ^
— 4 I KD
— 3 4 K D
— 2 5 K D
— 20KD
— I 7KD
I 4.4 -
— I 3 K D
— I 2 K D
Figure 5-7 Electrophoretic separation of cyanogen bromide
peptides of S5. The peptides were generated in tne presence
of 70% formic acid and CNBR as described in Methods. The
resulting peptides were resolved by 15% SDS-PAGE and
electroblotted onto PVDF membrane. The peptides were
visualized by coomassie staining and the peptide bands excised
and analyzed by gas phase chemical amino acid sequencing as
shown in table 5-3. The molecular weight markers are the same
as shown in figure 3-2.

155
TABLE 5-2
Summary of Ribosomal Proteins and Peptides Submitted for
Gas Phase Amino Acid Sequence Analysis
Protein
or
PeDtide
Preparation for
Sequencinga
Number of
residues
Seauenced
Repetitive
Yield
m
Amount
initial-
AA (Dmol)
S4
Spotted onto PVDF
30
96.1
Leu 136
S8
Bathed in eluant
20
92.9
Trp? 5
S25
Spotted onto PVDF
34
97.2
Lys 200
S5
Electroblotted
0
-
-
S5
Electroblotted0
0
—
—
CNBR Peptides of S5:
25 kDa
Electroblotted
36
94.1
Val 11
12 kDa
Electroblotted
37
89.4
Arg 16
13 kDa
Electroblotted
37
90.5
Val 31
aSee Methods for experimental details. S5 and CNBR peptides
of S5 were resolved on 12 and 15% Laemmli polyacrylamide gelr
respectively and electroblotted onto PVDF membranes as
described in Methods.
bFollowing 5 sequencing cycles the protein content remaining
on the PVDF membrane was hydrolysed in HC1 and analyzed by
amino acid analysis and 17 pmol of S5 remained on the
membrane.
cFollowing electroblotting of S5 onto PVDF membrane, one third
of the membrane containing the blotted S5 protein was cut off
and analyzed for protein content by AA analysis. Based upon
the amino acid analysis 20 pmol of protein was on the membrane
submitted for sequence analysis.
peak was detected eluting just prior to dithiothreitol (DTT)
in the chromatogram (figure 5-8 A). It was suspected that
this peak was an oxidized product of Tryptophane, as reported
by Walsh et al. (1988), but the peak we observed eluted much
later, and due to differences in chromatography methods we
were not able to make an assignment for residue 5. Since a

156
TABLE 5-3
Summary of Amino Acid Sequence for Several Bovine
Mitochondrial Ribosomal Proteins and CNBR Peptides of S5
Protein Amino Acid Sequence
S4 H.N-L-L-S-A-A-Y-E-D-S-R-K-W-E-A-R-A-K-E-D-S-H-L-A-
D-A-A-X-X-M-H-
S8 h2n-w?-s-e-a-e-s-g-s-p-k-i-k-k-p-t?-f-m-d?-e-e-v?-
S25 H.N-K-N-R-A-A-R-V-R-V-G-K-G-N-K-P-V-T-Y-E-E-A-H-A-
P-P?-Y-I-A-H?-R-K-G-G?-L-
CNBR Peptides of S5:
25 kDa -V-R-K-P-A-L-E-L-L-H-Y-L-K-N-T-N-F-A-H-P-A-V-R-
Y-V-L-Y-G-E-K-G-T-G-K-T-L-
34 kDa and 17 kDa CNBR peptides have the same sequence as the
25 kDa peptide.
12 kDa -R-V-R-N-A-T-D-A-V-G-I-V-L-K-E-L-K-R-Q-S-S-L-G-
V-F-R-L-L-V-A-V-D-G-V-N-A-L-
20 kDa has the same sequence as the 12 kDa peptide.
13 kDa -V-K-N-D-W-Q-G-G-A-I-V-L-T-V-S-Q-T-G-S-L-F-K-P-
R-K-A-Y-L-P-Q-E-L-L-G-K-E-G-
a0ne letter code for amino acids used in this figure
number of oxidized products of Tryptophan exist, an empirical
approach was used in an attempt to determine if residue 5 was
indeed PTH-Tryptophane.
A test protein of known amino acid sequence (containing
Trp) was subjected to CNBR digestion and blotting onto PVDF in
a manner identical to that used for CNBR peptides of S5. This
was done to determine if Trp residues are lost and if so were

157
oxidized products of Trp present to indicate the presence of
Trp. Sperm whale myoglobin was used as a test protein because
its amino acid sequence was known and it has a Trp residue
near its amino-terminus (residue #7), to reduce the number of
cycles required to sequence before encountering a PTH-Trp
residue. In one treatment, 600 pmol of myoglobin was digested
by CNBR and the second sample of the same amount of myoglobin,
was not digested. Both samples were blotted onto PVDF in the
same manner as described for S5 and submitted for AA-sequence
analysis. Sequence analysis of residue 7 from undigested
myoglobin revealed 2.5 pmol of PTH-Trp and no new peak (figure
5-8 C) observed just prior to DTT as observed in residue 5 of
the CNBR peptide of S5 (Figure 5-7). This amount of PTH-Trp
was about half of the amount of other PTH-AA found in that
analysis. The large loss of material was due to losses in
transfer of protein to PVDF. However AA-sequence analysis of
this same residue following CNBR digestion revealed that PTH-
Trp was not found in amounts above background and the putative
oxidized PTH-Trp peak appeared at the same retention time,
just prior to the DTT peak (figure 5-8 B), as observed in the
sequencing of the 13 kDa CNBR peptide of S5. This suggests
strongly that Trp residues do not survive CNBR cleavage, but
under the conditions used in this study do survive electro¬
phoresis and electroblotting. Though these results do not
agree with the observations made by Matsudaira (1987), the
full extent of differences in electrophoretic and blotting
methods used could not be determined from the publication.

158
O P T U
B
6.4k D2L CNBR Peptide SW MYOGLOBIN
SW MYOGLOBIN
R e s * 7
Mi
n
i
w
^LJuñA.—
Figure 5-8 Elucidation of a putative oxidized tryptophan
residue found in the 5 PTH amino acid residue of the 13 kDa
CNBR peptide of S5. This was done by comparing the PTH
tryptophan residue #7 of sperm whale myoglobin which was
chemically digested by CNBR (panel B) in an identical manner
as S5 (panel A) and with no chemical digestion (panel C). All
three panels are HPLC chromatograms of PTH amino acid residues
resulting from Edman degradation chemistry of the respective
peptides and proteins. In all cases PTH analysis of residues
was done using an IBI 470 gas phase amino acid sequencer with
a model 120 A on-line PTH-HPLl analyzer. The arrows indicate
the presence of either oxidized tryptophan (panels A & B) or
tryptophan PTH residues (panel C). For additional
experimental details see Methods.

159
One likely difference that may account for the
observation was that in the electrophoresis of Laemmli gels,
0.1 mM thioglycolate was added to the cathode run buffer in
our method and not in the method reported by Matsudaira
(1987). Thioglycolate functions as a free radical scavenger
to reduce the possibility of N-terminal blockage and chemical
modifications to other residues and presumably protected Trp
residues during electrophoresis and blotting procedures.
The presence of the oxidized PTH-Trp residue, just prior
to the DTT peak, in CNBR peptides of Sperm Whale myoglobin and
13 kDa peptide of S5 strongly suggest that residue 5 in the
peptide of S5 is PTH-Trp.
Pi scussion
I have identified the GTP-binding protein in bovine
mitochondrial ribosomes. This finding is without precedent in
other ribosome systems. Procedures and strategies were
developed for the purification and microsequencing of S5 in
order to gain insight into the possible functional role of
this protein on the small subunit of mitochondrial ribosomes.
Additionally the primary sequence of mammalian mitochondrial
ribosomal proteins, which have never been reported in the
literature, are presented here. These sequences are partial
amino-terminal sequences of several mitochondrial ribosomal
proteins, as well as primary sequences from CNBR generated
peptides of S5 (Table 5-3).

160
Approximately one-third of the primary sequence of S5 has
been determined and two limited sequences discovered (GX^GKT
and DAVGIVL) probably contribute to the GTP binding domain in
S5, by virtue of their homology to corresponding sequences in
other GTP-binding proteins. These two sequences are
homologous to sequences found in the A and E sites which bind
to the phosphate side chain and the guanine base, respectively
(Figure 5-9). The sequence (GX4GKT) found in the 25 kDa CNBR
peptide of S5 is identical to that same limited sequence found
(A site) to interact with the a and r phosphates of GTP in all
GTP-binding proteins (table 5-4).
Residues in the E site are not as highly conserved as
those in the A site in all GTP binding proteins; nonetheless,
if conserved substitutions (Dayhoff, 1978) are allowed, this
limited sequence found in S5 (found in the amino terminus of
the 12 and 20 kDa CNBR peptides, Table 5-3) is homologous to
the corresponding sequence found in the E site of other GTP-
binding proteins as shown in Table 5-4. Though this
comparison was made to those GTP-binding proteins that
interact with ribosomes and to a limited number of residues,
they were to sequences that have been found to be highly
conserved and demonstrated to be functionally important in
their interactions with GTP. A computer search using the
entire known sequence for S5 CNBR peptides and other ribosomal
proteins (S8, S4, and S25) showed no significant homologies
with any other proteins in the GENEBANK data bank.

161
GX,GKT
S5
J 17kDa
J 25kDa
J 34kDa
DAVGXVL
1 12kDa
â– 
1
] 20JcDa
Ta rod [
GX,GKS
\ /
IF-2
LWN ~l 13kDa
EGVTCII NKXD
EF-Tu^.
A
C E
G
GX^GKT
DIWLW
NKXD
i
â– 
i
<( 1
GX^GKT DGAILW NKXD
v ✓ ^— / \ /
A
C E
G
m â–  â–  i
l
A C E G
GX.GKT DGAILW
s / . 1
NKXD
1 TT * â– 
â– 
â–¡
A C E
1.1 j
G
i 1
i i
. i
i
i )) 1
1
100
200
300
400
500
900
AMINO ACIDS
Figure 5-9 Comparison of putative conserved regions found in
the amino acid sequence of CNBR peptides of S5 to homolpgous
regions found in other GTP-binding proteins. The putative A
site and E site which have been shown to interact with the
pyrophosphate and guanine base of GDP respectively in other
GTP-binding proteins are shown for S5 and the corresponding
regions in other known GTP-binding proteins. For references
see figure 1-1. A putative peptide map for S5 is shown based
on overlapping sequence found among the CNBR peptides their
size and tne total molecular weight of S5. Portions of the
CNBR peptides that have been sequenced by chemical means are
shown as hash marked areas.

162
Table 5-4
ComDarison
of Amino
Acid Seauence
found in Mi toribosomal
Protein S5
with those
corresoondina
to the E site found in
other GTP-bindina Droteins involved
in Protein Svnthesis
Proteina
E Siteb
Homol oavc
S5
D-A-V-G-I-V-L
IF2
D-I-V-V-L-V-V
71%
EF-Tu
D-G-A-I-L-V-V
71%
EF-G
D-G-A-V-M-V-Y
57%
EF-Tumt
D-G-A-I-L-V-V
71%
a The source of these proteins are: S5, Bovine
mitochondrial; IF2, EF-Tu, and EF-G, E.
col i: EF-Tu t S. cerevisiae mitochondrial.
See figure 1-1 for references.
b Conserved substitutions (Dayhoff, 1978) were
allowed in making comparisons. Underlined
residues (one letter code) are those differing
from the sequence in S5. For a more
exhaustive comparison refer to figure 1-1 (E
site). Dayhoff (1978) conservative categories
are as follows: t; S,T,P,A,G; N,D,E,Q; H,R,K;
M,I,L,V; F,Y,W.
c The percent homology of amino acid sequence of
S5 are compared to the corresponding
region in other GTP-binding proteins shown
above. Conserved substitutions were allowed as
described in .

CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS
Mammalian mitochondrial ribosomes are known to differ
from other procaryotic, eucaryotic cytoplasmic, and even other
mitochondrial ribosomal systems in translocase specificity and
recognition of mRNA. O'Brien et al. (1990) discovered yet
another unusual characteristic of this ribosome, binding of
GTP directly to an integral site on the mitochondrial
ribosome. In this study I have further characterized this
high affinity binding and found that it is specific for GTP
and GDP. We now have an understanding of the minimal
molecular characteristics required for high affinity binding
by comparing binding affinities of GTP for the 28S ribosome to
other closely related nucleotides such as ITP, GMP, and 8-
Azido GTP, which bind to the ribosome with reduced affinity,
and other nucleotides (ATP, CTP, UTP, and periodate cleaved
GTP) which do not bind at all. This high affinity binding
could tolerate high salt concentrations, implying the binding
is not electrostatic in nature and persists at salt concen¬
trations that strips the ribosome of putative translocases and
even to the point where the integrity of the ribosome begins
163

164
to be lost (O'Brien, et al., 1990) suggesting strongly that
GTP binds directly to the 28S ribosome.
Probing deeper into this binding phenomenon, we
identified the protein that binds GTP on the ribosome to be
S5. This was accomplished by photochemically labeling the GTP
binding site on the ribosome with a photoreactive analog of
GTP and GTP itself. Both ligands specifically radiolabeled S5
following photoactivation by uv irradiation of the bound
ligand on the 28S ribosome. Ligand specificity for the
photoaffinity labeling of this ribosome was consistent with
the results of filter binding assays, strongly suggesting that
S5 specifically bound only GTP and GDP.
Micro methods were developed to purify, identify and
chemically sequence not only S5, but other mitochondrial
ribosomal proteins as well. Though none of these proteins
were shown to have any significant overall homologies to other
ribosomal proteins in other systems, strong homologies were
found to two sites of 7 to 8 residues in S5 to conserved areas
found in all GTP-binding proteins. The first of these
sequences found in S5, GX4GKT, is known to interact with the
pyrophosphate group of GDP and involved in GTPase activity in
other GTP-binding proteins. The other sequence, DAVGIVL, was
found to be homologous to sequences that interact with the
guanine base in GTP-binding proteins. Though only a third of
the S5 protein has been sequenced so far the identification of
at least two of the four highly conserved regions found in

165
GTP-binding proteins suggests that GTP binding in S5 may be
similar in its molecular detail as in other GTP-binding
proteins.
Efforts to find and clone the gene for S5 are in
progress. A number of approaches to obtaining this gene are
being pursued. Screening of cDNA expression libraries from
bovine liver and kidney and human hepatoma cells with the anti
S5 monospecific antibody, and probe screening a cDNA library
with redundant oligonucleotides complementary to the known
amino acid sequence of S5. An attempt is being made to
amplifying the S5 gene, using redundant oligonucleotide
primers also complementary to the known amino acid sequence of
S5, by the polymerase chain reaction from the above cDNA or
genomic libraries. This would provide not only the first
complete sequence of a mammalian mitochondrial ribosomal gene
to compare to other ribosomal proteins, but allow for a
complete comparison of S5 to other GTP-binding proteins.
The function of this GTP/GDP binding activity is unknown.
However, the fact that it binds to the small subunit leads one
to speculate that it is involved in protein synthesis
initiation complex formation, or possibly acting as a guanine
nucleotide exchange factor.
One possibility is that S5 is a resident IF2 like
molecule. In support of this hypothesis, it is known that
procaryotic IF2 forms a ternary complex with GTP, and
ami noacyl tRNA, on the 30S ribosome. The order of binding for

166
mRNA, tRNA, IF2, and 30S ribosome to form a competent
initiation complex is unimportant (Guaríerzi, 1986). In
contrast, in eucaryotic cytoplasmic ribosomes the binding of
the above components to eIF2 is tightly controlled (Dholakia
and Wahba, 1989) and a guanine exchange factor (GEF) mediates
transfer of the GTP/GDP molecule to the eIF2 prior to binding
the 40S ribosome to form the initiation complex. In addition
to GEF a number of accessory proteins exist to control
initiation complex formation and ultimately protein synthesis.
In mitochondrial ribosomal systems this degree of control over
protein synthesis may be even more relaxed. This would also
coincide with the fact that mitochondrial ribosome contain
nearly twice the number of proteins as procaryotic ribosomes
implying that at least some of these additional proteins may
have functional roles in protein synthesis. Studies are
underway to determine if mRNA and or mt-initiator tRNA
interact with S5 on the ribosome which would help substantiate
the above hypothesis.

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Yuen, S., Hunkapiller, M., Wilson, K., and Yuan, P. (1986),
SDS-PAGE Electroblotting. Add!ied Biosvstems User Bulletin
#25. Applied Biosystems, Foster City, CA.

BIOGRAPHICAL SKETCH
John Claude Anders was born April 21, 1951, in Miami,
Florida. He earned his Bachelor of Science degree in December
of 1974, majoring in biological sciences at Florida State
University. He also was married to Valerie M. McGee in August
of the same year. In December of 1978 he earned a Master of
Science in toxicology from Auburn University and also was
commissioned as a second lieutenant in the Alabama Army
National Guard in July of that same year. He accepted an
active duty commission as a first lieutenant in the Medical
Service Corp of the U.S.Army in November of 1978 and became a
member of the Department of Pharmacology in the Division of
Experimental Therapeutics at the Walter Reed Army Institute of
Research. There he studied the biotransformation and toxicity
of various experimental antimalarial and antileishmanial drugs
being developed for human use. In June of 1985, he continued
his graduate education in the Department of Biochemistry and
Molecular Biology at the University of Florida, Gainesville,
working under the direction of Dr. Thomas W. O’Brien.
Presently Major Anders is a principal investigator in the
Department of Immunology at the Walter Reed Army Institute of
Research, Washington D.C. He is working with a group that is
attempting to develop a malarial vaccine for use in humans.
179

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Thomas W. O'Brien, Chairman
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
ÚLa hi
Charles M. Allen, Jr. *
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
jl ¿JL
Robert -J. Cohen
Associ ate Professor of Bi ochemi stry
and Molecular Biology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
w
Ben M. Dunn
Professor of Biochemistry and
Molecular Biology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of/Phi 1osophy.
—
Wi Hi am W. Hauswi rth
Professor of Immunology
and Medical Microbiology
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirement for the
degree of Doctor of Philosophy.
December, 1990
Dean, Graduate School

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