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INTRODUCTION |
The translational elongation factors EF-1
(eEF1A)1 and EF-Tu (EF1A) are
GTP-binding proteins that serve similar roles in protein synthesis in
prokaryotes and eukaryotes, respectively. Their normal role is to
deliver aminoacylated tRNAs into the A site of the ribosome. While
dispensing this function for tRNAs carrying all the standard 20 amino
acids (including methionine) that are elongationally inserted into
proteins, elongation factors must discriminate against non-aminoacylated tRNAs and against the methionylated initiator tRNAMet that is loaded into the ribosomal P site by
initiation factors.
The basis for these functions is well understood in bacterial systems,
for which extensive ligand binding and structural studies have been
performed. The reported equilibrium dissociation constants for the
interactions of Escherichia coli EF-Tu·GTP with
aminoacylated elongator tRNAs fall between 0.2 and 7 nM
(1-3), varying over a 13.8-fold (4) or a 34-fold (2) range when
determined in a single set of experiments. These tight associations
forming aminoacyl-tRNA·EF-Tu·GTP ternary complexes contrast
strongly with the weak affinity of E. coli EF-Tu·GTP for
uncharged tRNA (Kd = 2.6-2.8 µM; Ref.
3). The 2250-fold weaker affinity for tRNAPhe lacking the
esterified phenylalanyl moiety (3) effectively prevents uncharged tRNA
from reaching the ribosomal A site unless the normal aminoacylation
status of cellular tRNAs is severely impaired. A considerably smaller
binding differential exists in the case of methionylated initiator
tRNAMet; the Kd values for binding
elongator methionyl-tRNAMet and initiator
formylmethionyl-tRNAMet were reported as 1.8 and 136 nM, respectively (3), a 76-fold difference. Exclusion of
initiator formylmethionyl-tRNAMet from the A site thus
relies only partially on EF-Tu·GTP discrimination, but also on A site
competition by elongator methionyl-tRNAMet·EF-Tu·GTP
ternary complex, complex formation with IF2·GTP, and selective
interaction of initiator tRNA with the ribosomal P site (5).
The recently solved crystal structure of the
phenylalanyl-tRNAPhe·EF-Tu·GTP complex from
Thermus aquaticus (6) has shown that the protein contacts
the aminoacyl-tRNA only in the acceptor/T half of the tRNA molecule.
Accordingly, aminoacylated tRNA half-molecules and tRNAs lacking the
anticodon domain have been reported to bind Thermus
thermophilus EF-Tu·GTP with affinities similar to those of the
parental tRNAs (7, 8).
The overall similarity of EF-1
function to that of EF-Tu (see,
e.g., Refs. 9-11) has been convincingly established by the interchangeability of EF-Tu and EF-1
in binding to, although not in
supporting protein synthesis by, bacterial and mammalian ribosomes
(12). EF-1
, like EF-Tu, has been shown to form ternary complexes
with GTP and aminoacyl-tRNA (9, 10, 13). However, in contrast to the
rich information available for EF-Tu, quantitative data on the
interaction of EF-1
with tRNAs are almost non-existent. The only
estimate for the stability of aminoacyl-tRNA·EF-1
·GTP ternary
complex we have found in the literature is one of "about 10 nM" for Phe-tRNA (11), and the extent of binding
discrimination against uncharged tRNA is not known. With regard to the
exclusion of initiator methionyl-tRNAMet from the A site of
eukaryotic ribosomes, it has been shown that a 2'-phosphoribosyl
modification of the purine at position 64 of the yeast and wheat germ
initiator tRNAs is an important antideterminant of interaction with
EF-1
·GTP (14, 15). After removal of this modification, both tRNAs
could serve as elongators in in vitro protein synthesis
(15); in the yeast case, the demodified methionyl-tRNA showed an
increased ability to enter ternary complex with EF-1
·GTP (14),
although no dissociation constants were reported.
In order to improve our understanding of the detailed function of
EF-1
in eukaryotic protein synthesis, we have studied the binding of
wheat germ EF-1
·GTP to various charged and uncharged RNAs. Our
results emphasize the similarity in the RNA binding properties of
higher eukaryotic EF-1
·GTP to those of its bacterial homologue
EF-Tu·GTP.
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EXPERIMENTAL PROCEDURES |
Preparation of RNAs--
Mature tRNAPhe from
E. coli strain MRE600 was purchased from Boehringer
Mannheim, total wheat germ tRNA was purchased from Sigma, and purified
yeast initiator tRNAMet was a gift from Drs. C. Florentz
and R. Giegé (Institut de Biologie Moléculaire et
Cellulaire, Strasbourg, France).
Mature tRNAVal, and initiator and elongator
tRNAsMet were purified from total wheat germ tRNA by
hybridization affinity using a procedure based on those of Tsurui
et al. (16) and Mörl et al. (17).
3'-Biotinylated deoxyoligonucleotides complementary to the 30 nucleotides upstream and including the discriminator base (nucleotide
73) were synthesized by automated chemistry; these oligomers were based
on the sequences of higher plant tRNAVal (lupine; Ref. 18),
elongator tRNAMet (wheat germ; Ref. 19), and initiator
tRNAMet (wheat germ; Ref. 20). About 15 µg of each tRNA
was obtained from 4 mg of total tRNA. Purity was assessed by
3'-labeling with [5'-32P]cytidine bisphosphate and T4 RNA
ligase (21) and analysis of ribonuclease T1 digestion products by 20%
sequencing polyacrylamide gel electrophoresis; no products apart from
those expected from the selected sequence were observed (data not shown).
All other tRNAs were generated in vitro by transcription
with T7 RNA polymerase in the presence of 10 mM 5'-GMP and
1 mM GTP to produce 5'-monophosphate termini (22). RNAs
were purified by denaturing polyacrylamide gel electrophoresis (8%),
recovered by electroelution, and dialyzed against water.
Transcriptional templates were either plasmid DNAs linearized at a
BstNI restriction site coincident with the 3'-CCA, or DNA
amplified in vitro by polymerase chain reaction (23).
Plasmid templates were used to make unmodified lupine
tRNAVal (synthetic clone pTVAL, which encodes
tRNAVal with a CAC anticodon in place of the modified IAC
(I = inosine) of the mature tRNA), E. coli
tRNAAla and short derivatives thereof (8), and wheat germ
elongator tRNAMet (24).
TYSma RNA, a 3'-fragment of turnip yellow mosaic virus
(TYMV)2 RNA, was made by
transcription with T7 RNA polymerase from BstNI-linearized plasmid DNA as described (25).
Preparation of Aminoacylated RNAs--
RNAs (30-50 pmol) were
preparatively aminoacylated with appropriate radiolabeled amino acids
and aminoacyl-tRNA synthetases. The completed reactions were acidified
by adjusting to 75 mM sodium acetete (pH 5.2),
deproteinized with phenol/chloroform equilibrated to pH 5.2 with sodium
acetate solution, and ethanol-precipitated. After recovery by
centrifugation, the valylated RNAs were redissolved in 5 mM
sodium acetate (pH 5.2) and stored at
80 °C until use. The plant
tRNAs, yeast initiator tRNAMet, and the viral TYSma RNA
were aminoacylated with activities present in a partially purified
wheat germ extract (26), in TM buffer (25 mM Tris-HCl (pH
8.0), 2 mM MgCl2, 1 mM ATP, 0.1 mM spermine) containing 10 µM
[3H]valine (47.2 Ci/mmol), 10 µM
[3H]alanine (50 Ci/mmol), or 10 µM
[35S]methionine (350-1200 Ci/mmol) at 30 °C. E. coli tRNAPhe was aminoacylated with
[3H]phenylalanine (136 Ci/mmol), and E. coli
tRNAAla and its short derivatives were aminoacylated with
[3H]alanine (50 Ci/mmol) using purified recombinant
E. coli aminoacyl-tRNA synthetases as described (8).
EF-1
·GTP Binding Assays--
Purified wheat germ EF-1
(27) was activated by incubation with 20 µM GTP in EF
buffer (40 mM HEPES (pH 7.5), 100 mM
NH4Cl, 10 mM MgCl2, 1 mM dithiothreitol) plus 25% (v/v) glycerol at 30 °C for
20 min. Various concentrations (0-250 nM) of EF-1
·GTP were incubated together in multiwell strips (Nunc) with valylated RNAs
(0.2-5 nM) in EF buffer containing 12.5% glycerol and 0.5 mg/ml fragmented salmon sperm DNA on ice for 15 min (after Ref. 13) in
20 µl reactions. The amounts of ternary complex were estimated with a
ribonuclease protection/ trichloroacetic acid filter precipitation
assay adapted from Louie et al. (4). Incubations were
terminated by addition of 4 µl of ice-cold 5 mg/ml ribonuclease A
(Sigma) and incubation for 15 s before quenching the ribonuclease activity with the further addition of 4 µl of 50 mg/ml tRNA or total
torula RNA (Sigma). Total reaction mixtures were then immediately pipetted onto dried 1-cm filter paper squares (3MM, Whatman)
impregnated with 20% trichloroacetic acid containing 2 mM
of the appropriate amino acid, and immediately plunged into excess 10%
trichloroacetic acid to be held on ice for at least 5 min. After
several washes with 5% trichloroacetic acid and 95% ethanol, the
dried filters were subjected to counting in a liquid scintillation spectrometer.
Equilibrium dissociation constants (Kd) were
calculated from binding curves comprising data from 14 concentrations of EF-1
·GTP. Each binding assay was performed in duplicate, and repeated at least twice. The concentration of active EF-1
·GTP present in binding experiments was determined by binding in the presence of excess [3H]Val-tRNAVal
transcript. Aminoacyl-tRNA·EF-1
·GTP ternary complex formation follows Equation 1.
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(Eq. 1)
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In terms of total concentrations of aminoacylated tRNA
(t) and EF-1
·GTP (e), and defining the
concentration of ternary complex as c, this equation becomes
Kd = ((t
c)(e
c))/c, which
can be rearranged to c2
c(t + e + Kd) + t·e = 0. Solution for c, the
quantity assayed in the binding reaction, by means of the general
quadratic equation yields c = ((t + e + Kd) ±
((t + e + Kd)2
4t·e))/2.
The plotting program KaleidaGraph (Adelbeck Software) was used to solve
the plot of c (filter cpm) versus e,
yielding Kd, t, and the y
intercept representing the level of background counts. Note that, in
practice, t (total concentration of charged tRNA competent
to enter ternary complex) is an unknown quantity, since not all the
aminoacyl-tRNA is capable of forming ternary complexes, presumably due
to denaturation. Typically, 80-95% of aminoacylated tRNAs were bound,
but in some instances lower proportions of charged tRNAs were competent
for binding (see Tables II and III).
The plotted equation incorporated a specific activity constant
converting tc filter cpm to moles. This specific activity
constant was determined by assaying 3H- or
35S-labeled aminoacylated tRNA immediately after desalting
with a Sephadex G50 spin column. Counts (cpm) from direct counting in
scintillation fluid at known efficiency were compared with counts
obtained after taking an aliquot of the labeled tRNA in 20 µl of
binding buffer through a mock assay termination procedure (including
addition of RNA and spotting onto filter paper). This specific activity
measurement thus included adjustment for losses during transfer to the
filter paper and from incomplete precipitation onto the paper.
Competition Binding Assays--
Assays were performed as above,
except that all EF-1
·GTP concentrations were represented in
triplicate, and two binding curves for the
[35S]methionyl-tRNAMet transcript were
performed in parallel in the presence and absence of unlabeled
competitor RNA. The same quadratic function used for fitting standard
binding curves gave a good fit to the data determined in the presence
of competitor RNA (similar chi-square value), and was used as an
approximation of the extremely complex function describing the
competition. The difference in Kd estimated in the
presence and absence of competitor ("Kd offset"), which represents the concentration of competitor
RNA·EF-1
·GTP complex when the reporter is half-bound, was then
used to calculate the Kd for the competitor RNA.
Equation 1 applied to the competitor RNA is shown by Equation 1a; when
the reporter is half-bound, [free EF-1
·GTP] = Kd (reporter), this latter term being determined in
the binding assay lacking competitor.
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(Eq. 1a)
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Thus, the above equation becomes Equation 2.
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(Eq. 2)
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The competition binding assay was compared with the direct assay
by determining the Kd for a
[3H]valylated viral RNA both ways (data not shown). The
direct assay yielded Kd = 2.9 ± 0.5 nM, while the competition assay yielded
Kd values of 1.5 and 2.0 nM in two runs.
The similarity of these estimates validates the competition assay.
De-modification of Initiator tRNAMet--
Periodate
oxidation and ribose removal by
-elimination were performed on wheat
germ and yeast initiator tRNAMet as described (15), except
that the reaction products were not subjected to chromatographic
purification. The treatment results in loss of the phosphoribosyl
modification of nucleotide 64 as well as removal of the 3' nucleotide.
Demodification was judged complete, since the 3' nucleotides were shown
after 3'-labeling to have been quantitatively removed. The treated
tRNAs were methionylated by a wheat germ methionyl-tRNA synthetase
activity in the presence of (CTP,ATP):tRNA nucleotidyltransferase,
which is able to regenerate the eliminated 3'-A residue.
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RESULTS |
Equilibrium Dissociation Constants for Aminoacylated tRNAs--
A
ribonuclease protection assay based on that described for the EF-Tu
studies of Louie et al. (4) was developed to measure the
dissociation constants for the interactions between EF-1
·GTP and
various aminoacylated tRNAs. This assay relies on the protection of the
RNA bearing an amino acid labeled with 3H or
35S against ribonucleolytic degradation that occurs when
the RNA is bound by EF-1
. To make the technique more convenient,
cheaper, and more accessible to RNAs available in limited quantities,
the binding reactions were reduced in size to 20 µl. This permitted the entire reaction to be spotted onto paper filters at the end of the
assay, avoiding cumbersome filtration. After treatment with the high
levels of ribonuclease A necessary to digest unprotected phosphodiester
bonds within 15 s on ice, rapid quenching of the ribonuclease was
needed to prevent further digestion while the RNA was precipitated onto
filter paper squares. This was achieved by addition of ice-cold RNA,
rapid spotting onto filter paper that had been dried after impregnation
with 20% trichloroacetic acid, and immediate addition to a 10%
trichloroacetic acid bath held on ice. To permit binding assays at
subnanomolar concentrations of aminoacylated tRNA, necessary when using
[35S]methionyl-tRNAMet as a reporter in
competition binding assays (see below), fragmented salmon sperm DNA
(0.5 mg/ml) was included in all binding reactions in order to prevent
adsorptive losses of labeled RNA to the plastic walls of the incubation vessel.
Representative binding curves of four of the aminoacylated RNAs studied
are shown in Fig. 1, and the dissociation
constants determined from at least three replicates are reported in
Table I. The tRNAs assayed were of four
specificities (valine, methionine, alanine, and phenylalanine), and
included tRNAs of higher plant or bacterial origin; the tRNAs were
either fully modified mature tRNAs purified from cells, or in
vitro transcripts lacking post-transcriptional modifications but
with the natural 5'-GMP termini. The dissociation constants measured
for the aminoacylated tRNAs varied between about 1 and 10 nM. Only small differences in binding affinity were
observed between the fully modified and unmodified forms of plant
tRNAVal and tRNAMet (both forms were derived
from the same gene in the case of tRNAMet). No significant
difference was observed between the binding affinities of the plant
(Arabidopsis thaliana) and bacterial (E. coli)
tRNAAla transcripts (5.3 versus 6.5 nM). Both alanyl-tRNA and phenylalanyl-tRNA were bound
considerably less tightly than valyl-tRNA and methionyl-tRNA.

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Fig. 1.
Representative binding curves for four RNAs
listed in Table I. A, binding curve for
[3H]valyl-tRNAVal, present at 4.46 nM; Kd was estimated as 0.88 nM. B, binding curve for
[3H]valyl-tRNAVal transcript, present at 4.12 nM; Kd was estimated as 2.44 nM. C, binding curve for
[3H]phenylalanyl-tRNAPhe, present at 5.28 nM; Kd was estimated as 10.1 nM. The specific activities of [3H]valine and
[3H]phenylalanine used to fit the curves shown
(KaleidaGraph) were 492 and 1449 filter cpm per nM
concentration in the 20-µl binding reaction, respectively.
D, binding curve for [3H]valyl-TYSma RNA,
present at 5.28 nM; Kd was estimated as
2.00 nM.
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The rather narrow range in the dissociation constants of the
aminoacylated tRNAs studied in Table I (12-fold) indicates a capacity
for wheat germ EF-1
·GTP to form tight complexes with tRNAs of
widely differing sequences. Despite varying primary sequence, however,
tRNAs all share very similar L-shaped tertiary conformations. Different
structural motifs (a pseudoknotted amino acid acceptor stem; Refs.
28-30) are found in some aminoacylatable plant viral RNAs that have
been shown to interact with EF-1
(29, 31), although dissociation
constants for these interactions have never been reported. TYMV RNA has
a 3'-terminal 82-nucleotide-long tRNA-like structure that can be
aminoacylated with valine (26, 32). To assess the affinity of EF-1
for pseudoknotted RNA, we prepared 264-nucleotide-long TYSma
transcripts (25) that included the tRNA-like structure shown in Fig.
2, and assayed the binding affinity of
the valylated RNA to wheat germ EF-1
·GTP. The dissociation constant (1.9 nM; Table I) measured for the valylated viral
RNA was comparable to that of the unmodified valyl-tRNAVal
transcript (2.3 nM), indicating a capacity for
EF-1
·GTP to form tight complexes with RNAs of varying acceptor
stem architecture. Tight complex formation was dependent on EF-1
activation and was not observed for EF-1
·GDP, and valylated viral
RNA competed with valylated tRNAVal for binding by
EF-1
·GTP (data not shown). These observations confirm that the
valylated viral RNA was bound by the same binding surface as
aminoacyl-tRNA.

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Fig. 2.
Comparative structures of two high affinity
ligands of EF-1 ·GTP. The structure marked
tRNAVal represents the unmodified transcripts of
A. thaliana tRNAVal; that marked TYMV
represents the 3'-82-nucleotide-long tRNA-like structure that is
present in TYSma transcripts. To assist in following the linear
sequence through the pseudoknotted amino acid acceptor stem, the TYMV
nucleotides are numbered (from the 3'-end). Loops
L1 and L2, which cross the major and minor
grooves of the helix, respectively (30), are indicated. Nucleotide 64 is arrowed on the tRNAVal structure to indicate
the position of the 2'-phosphoribosyl modification of initiator
tRNAMet (see "Results").
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Discrimination between Charged and Uncharged tRNA--
The high
specific activity labeling achievable for tRNA aminoacylated with
[35S]methionine made this an excellent reporter species
in a competition variant of the standard assay, permitting a study of
the affinity of wheat germ EF-1
·GTP for uncharged wheat germ tRNA.
The addition of 10 µM uncharged wheat germ tRNA to 0.47 nM [35S]methionyl-tRNAMet
transcript resulted in a slightly weakened apparent binding affinity of
the reporter methionyl-tRNAMet. The calculated
Kd of 15.2 ± 6.3 µM for the
uncharged tRNA (Table I) means that the affinity of EF-1
·GTP for
uncharged tRNA is 103-fold to
104-fold weaker than for charged tRNA.
Phosphoribosyl Modification of Initiator tRNAMet as an
Antideterminant of Interaction with EF-1
·GTP--
Mature
initiator tRNAMet and elongator tRNAMet were
purified by a hybridization affinity method from total wheat germ tRNA.
3'-Labeling with 32P revealed no detectable
cross-contamination of signature oligonucleotides generated by
ribonuclease T1 between these two tRNAMet preparations
(data not shown). In contrast to the tight binding of elongator
[35S]methionyl-tRNAMet (Kd = 0.83 nM; Table I), initiator
[35S]methionyl-tRNAMet bound weakly to
EF-1
·GTP. At accessible concentrations of EF-1
·GTP, the
binding curves were incomplete, preventing reliable estimation of the
dissociation constant. However, the shape of the curve suggests a
dissociation constant >100 nM (Table
II). Similarly weak binding was observed
to yeast initiator [35S]methionyl-tRNAMet
(data not shown).
Both the wheat germ and yeast initiator tRNAMet were
oxidized with periodate in order to remove the 2'-ribosyl modification
of nucleotide 64 by
-elimination (15, 33). The oxidized,
phosphatase-treated tRNAs were preparatively aminoacylated with
[35S]methionine using a methionyl-tRNA synthetase
activity from wheat germ that also contains (CTP,ATP):tRNA
nucleotidyltransferase. This latter activity replaced the 3'-terminal A
residue removed by the
-elimination procedure, permitting subsequent
aminoacylation. Both oxidized initiator methionyl-tRNAs formed high
affinity complexes with EF-1
, and produced binding curves similar to
those of elongator methionyl-tRNAMet, with
Kd values of 2.5 and 12 nM for the wheat
germ and yeast tRNAs, respectively (Table II). There was a consistently higher background when using the oxidized tRNA, and a rather low proportion of the oxidized methionyl-tRNAs was capable of forming ternary complex (33% and 34% for two different preparations of oxidized wheat germ tRNAMet); the latter was probably due
to incomplete removal of the phosphoribosyl group or other damage to
the molecule resulting from the chemical treatment. Nevertheless, these
results clearly show that the phosphoribosyl modification of nucleotide
64 is a powerful antideterminant of EF-1
·GTP binding.
EF-1
·GTP Interacts Primarily with the Acceptor/T Arm of
tRNA--
Three truncated derivatives of E. coli
tRNAAla that had previously been used in studies with EF-Tu
(8) were used to assess the part of tRNA interacting with wheat germ
EF-1
·GTP (Fig. 3). The
Anticodon
RNA has the 17-nucleotide anticodon stem/loop replaced with a UUAA
spacer. The Ala minihelix RNA lacks all D- and anticodon arm sequences,
with the remaining acceptor/T arm built as a continuous 12-base pair
helix. RNA 12 is an RNA that emerged from sequential in
vitro selections as a tight ligand of T. thermophilus
EF-Tu·GTP (8), and contains a 9-residue bulge loop interrupting the
Ala minihelix RNA. It was thought that the bulge mimicked a known bend
between the T and acceptor stems of tRNA (34), thereby improving the
stability of ternary complexes formed with charged minihelix-type
RNAs.

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Fig. 3.
The sequences and secondary structures of
RNAs based on E. coli tRNAAla used in binding
studies.
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Alanyl-
Anticodon RNA was bound by wheat germ EF-1
·GTP with a
Kd only about 3-fold higher than that for the
alanyl-tRNAAla transcript (Table
III), indicating that the anticodon
domain is not essential for binding. As observed in studies with
T. thermophilus EF-Tu·GTP, a rather low fraction of the
deleted alanyl-RNA formed ternary complexes (Table III), suggesting a
tendency for this RNA to assume a conformation not recognized by
EF-1
·GTP. Note that the alanylated transcripts of E. coli tRNAAla were themselves bound to only 56% in our
assays, considerably lower than the 80-95% typical of charged tRNAs.
Alanyl-RNA 12 was bound somewhat less tightly, with a
Kd 4.5 times higher than that of the
alanyl-tRNAAla transcripts, but some 3-fold tighter than
alanyl-Ala minihelix (Table III). Incomplete binding curves for this
latter RNA indicated poor interaction with EF-1
·GTP. These results
demonstrate that, as with EF-Tu, a bent acceptor/T arm is preferred for
binding, and that most of the binding affinity for aminoacyl-tRNA is
derived from interaction with the acceptor/T portion of the tRNA.
Residues Contacting Aminoacyl-tRNA Are Conserved between EF-Tu and
EF-1
--
The close similarity in RNA binding properties between
bacterial EF-Tu and wheat germ EF-1
prompted a detailed sequence
comparison of these proteins to determine whether sequence conservation
is especially high in regions of T. aquaticus EF-Tu that are
in contact with the aminoacyl-tRNA. The best studied EF-Tu proteins,
from E. coli, T. thermophilus, and T. aquaticus,
which are known to share the same folded structure (35, 36), each have
about 33% sequence identity and 55% weighted similarity in primary
sequence with wheat EF-1
.3
The sequence identity is scattered throughout the protein, permitting ready alignment of all EF-Tu and EF-1
sequences in the data base (Fig. 4). Secondary structure prediction
by the PHDsec program at the PredictProtein server (37) for T. aquaticus EF-Tu was in good agreement with the crystal structure
(6), and the secondary structure elements predicted for human and wheat
EF-1
could be readily fitted to the EF-Tu structure. Alignment based
on predicted structure violated no alignments of universally conserved
amino acids (data not shown). Higher eukaryotic EF-1
s differ by some 7 insertions and 3 deletions from EF-Tu. Mammalian and plant EF-1
s are 18% and 14% longer, respectively, than E. coli EF-Tu,
one of the shortest. Insertions or deletions are almost exclusively placed in the loops and at the N and C termini of the EF-Tu structure, rather than within helix or sheet structural elements (Fig. 4), such
that overall structure would be expected to be conserved.

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Fig. 4.
Sequence alignment between EF-Tu and
EF-1 . The EF-1 sequences from wheat, yeast, and humans
(accession numbers Q03033, P02994, and P04720, respectively) were
aligned with the EF-Tu sequences from T. aquaticus and
E. coli (accession numbers Q01698 and P02990, respectively).
Amino acids conserved between the two EF-Tu species shown and at least
90% of the 64 EF-1 sequences retrieved from the data base are
shaded. Also shown are sheet (b) and helix
(a) secondary structure elements for T. aquaticus
EF-Tu (6), and points of contact with GTP·Mg2+
(G; including Mg2+ contacts through water
molecules), the CCA-phenylalanyl moiety (C), the 5'-terminus
(5), and T-stem (T) of the bound aminoacyl-tRNA
(data taken from crystal structures in Refs. 6, 35, and 36). Domains 1, 2, and 3 of T. aquaticus EF-Tu have approximate boundaries
at amino acids 1-238, 239-310, and 310-405, respectively.
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In Fig. 4, highly conserved amino acids have been marked along with the
secondary structure elements and ligand contacts taken from the EF-Tu
ternary complex crystal structure (6). Amino acids that contact the
GTP·Mg2+ and aminoacyl-tRNA noticeably coincide with or
are close to clusters of conserved residues (Fig. 4). This is clearly
the case for most of the GTP·Mg2+ contacts (in domain 1)
and the contacts with the aminoacyl- and 5'-termini of the bound RNA
(in domains 1 and 2; Fig. 4). The interactions of T. aquaticus residues Tyr-47, Asp-51, and
Ser164-Ala165-Leu166 with
GTP·Mg2+, and of residues Lys52 with the
phosphates in the 3'-CCA of the tRNA (6), are an exception, although
these residues are close to a single conserved amino acid. Contacts
with the T-stem of the bound tRNA in domain 3 involve less conserved
amino acids, partly because domain 3 is considerably less conserved
than the other two domains. Nevertheless, the T-stem contacting
T. aquaticus residues, Gln341 and
Gly391, fall within clusters of conserved residues.
The general coincidence of ligand-contacting amino acids with conserved
residues means that the length heterogeneities and regions of low
sequence conservation are separated from the elements crucial for
ternary complex formation. The combined sequence and structure
alignment in Fig. 4 is consistent with the model that EF-1
has a
core structure close to that of EF-Tu, bearing superficial elaborations
mainly in external loops not in contact with aminoacyl-tRNA. This
interpretation is consistent with the closely similar RNA binding
properties of EF-Tu and EF-1
. Supportive experimental evidence comes
from cross-linking studies between rabbit EF-1
·GTP and
aminoacyl-tRNA (38) that indicate contact sites corresponding to
T. aquaticus EF-Tu residues 270-275, 345-355 and 375, sites that are in contact with the RNA in the ternary complex crystal (6). The rather large sequence variability surrounding the GTP·Mg2+ binding site (insertion in the helix near
T. aquaticus residue 35 to produce a predicted
helix-loop-helix, and variability in the helix between residues 144 and
161; Fig. 4) may be responsible for the different nucleotide binding
properties of EF-Tu and EF-1
. EF-1
has similar affinity for GTP
and GDP, while EF-Tu has a 100-fold higher affinity for GDP than for
GTP (13).
 |
DISCUSSION |
High Affinity Binding of EF-1
·GTP to Aminoacylated
tRNAs--
The equilibrium dissociation constants of ternary complexes
formed by wheat germ EF-1
·GTP with tRNAs of four specificities (valyl, methionyl, alanyl, and phenylalanyl) were between 0.8 and 10 nM (Table I), indicating a high affinity interaction
similar to that between E. coli EF-Tu·GTP and
aminoacyl-tRNAs (Kd values of 0.2-7 nM;
Refs. 1-3). Phenylalanyl-tRNAPhe from E. coli was bound by EF-1
·GTP with the weakest affinity of the
aminoacyl-tRNAs tested. Since there was no significant difference
between the binding of plant and E. coli
alanyl-tRNAAla transcripts to EF-1
·GTP (5.3 and 6.5 nM, respectively; Table I), the weaker binding of
Phe-tRNAPhe is unlikely to be due to the fact that this
tRNA and the EF-1
are from different sources. The 12-fold range of
Kd values we have observed for the
aminoacyl-tRNA·EF-1
·GTP complexes for a subset of the 20 amino
acid specificities falls within the 13.8-fold (4) and 34-fold (2)
ranges measured for aminoacyl-tRNA complexes with E. coli
EF-Tu·GTP. However, the relative affinities of different aminoacyl-tRNAs do not appear to strictly follow those observed for
EF-Tu (4). Mature wheat germ Met-tRNAMet and
Val-tRNAVal bound rabbit EF-1
·GTP (kindly provided by
Dr. W. Merrick) with Kd values of 1.2 ± 0.1 nM and 1.1 ± 0.3 nM, respectively (data
not shown); the same aminoacyl-tRNAs were bound with similar affinity
by wheat germ EF-1
·GTP (Kd values of 0.83 and 1.0 nM, respectively; Table I). The stabilities of ternary
complexes formed with higher eukaryotic EF-1
·GTP are thus clearly
similar to those formed by bacterial EF-Tu·GTP.
Only about a 2-fold difference in the binding affinities to wheat germ
EF-1
·GTP were observed between the fully modified and unmodified
forms of Val-tRNAVal and Met-tRNAMet (Table I).
Similar approximately 2-fold differences were observed between the
Kd values for EF-Tu·GTP ternary complexes containing modified and unmodified aminoacyl-tRNAs (aspartate, Ref. 7;
phenylalanine, Ref. 39). Thus, in both the eukaryotic and prokaryotic
systems, modified nucleotides, such as the thymine and pseudouracil
bases present in the T-loops of virtually all tRNAs, exert little or no
influence on ternary complex formation.
Binding Discrimination against Two Types of tRNAs That Are Normally
Excluded from the Ribosomal A Site--
Since the role of
EF-1
·GTP is to deliver aminoacyl-tRNAs to the ribosomal A site, it
was of interest to determine the extent to which two A site non-ligands
(uncharged tRNA and initiator Met-tRNAMet) are excluded
from the A-site by virtue of weak interaction with EF-1
·GTP.
Uncharged wheat germ tRNA interacted very weakly with EF-1
·GTP
(Table I). The 103-fold to 104-fold difference
in the Kd values of ternary complexes containing
charged and uncharged tRNAs is similar to the 2250-fold difference in
Kd values for the equivalent ternary complexes formed with E. coli EF-Tu·GTP (3). Thus, in both the
eukaryotic and prokaryotic systems, the poor ability to form ternary
complexes with EF·GTP is a major factor in excluding uncharged tRNA
from the ribosomal A site.
Discrimination at the level of ternary complex formation with
EF-1
·GTP is clearly also a factor in excluding initiator
Met-tRNAMet (Kd > 100 nM;
Table II; compare with Kd = 0.83 nM for
elongator Met-tRNAMet; Table I) from the A site. Although a
better estimate for the ternary complex Kd will need
to be obtained in the future, it appears that the role of
discrimination by EF-1
·GTP in plants is at least as significant as
that of EF-Tu·GTP discrimination in E. coli (the
Kd for the EF-Tu·GTP ternary complex with
initiator formylmethionyl-tRNAMet was reported as 136 nM; Ref. 3), but probably not as dominant as in the case of
uncharged tRNA. Removal from the wheat germ initiator tRNA of the
phosphoribosyl modification of nucleotide 64 that is a characteristic
of plant and yeast initiator tRNAs (33) led to high affinity binding by
EF-1
·GTP (Kd = 2.5 nM; Table I).
Since the ratio of this Kd to that for the ternary
complex with elongator Met-tRNAMet is only 3, it is evident
that the phosphoribosyl modification acts as a powerful antideterminant
blocking EF-1
·GTP binding. This modification is positioned at the
base of the T stem (see Fig. 2) in a position that is in close
proximity to the protein in the EF-Tu·GTP ternary complex (6). Our
results are in agreement with the experiments of Sprinzl and co-workers
(14, 15) on the critical role of modified nucleotide 64.
EF-1
·GTP Interacts Primarily with the Acceptor/T Half of
Aminoacyl-tRNA--
The relatively tight complexes formed between
EF-1
·GTP and two alanylated deletion variants derived from
tRNAAla (
Anticodon and 12 RNAs; Table III) show that
regions of the tRNA outside the acceptor/T arm play at most a minor
role in complex formation. Both alanyl-RNAs were far superior
EF-1
·GTP ligands to uncharged tRNA. The tighter ternary complex
formation of alanyl-12 over alanyl-Ala minihelix RNA (Table III), also
observed with T. thermophilus EF-Tu·GTP (8), suggests that
a kinked acceptor/T arm as revealed by x-ray crystallographic studies
for tRNAs (34) is more favorable for binding than a straight helix. An
uninterrupted acceptor/T helix may not readily undergo the
conformational change needed to assume the generalized conformation
that EF-Tu·GTP is suggested to impose on the various aminoacyl-tRNAs
on binding (3, 40). Interestingly, the high affinity binding to
valylated-TYSma RNA (Table I), which has a pseudoknotted acceptor stem
(Fig. 2), indicates that minor structural variations in the acceptor/T arm are compatible with tight binding to EF-1
·GTP. Perhaps the observed flexibility between the stacked helices of the TYMV acceptor/T arm (30) helps to make this RNA such an excellent ligand to EF-1
·GTP.
Our findings indicate strong similarities in the way that eukaryotic
and prokaryotic EF·GTPs interact with aminoacyl-tRNAs to form ternary
complex, supporting an overall structure of the EF-1
ternary complex
similar to that of T. aquaticus EF-Tu (6), rather than the
structure proposed by Kinzy et al. (38). The demonstrated
interaction of EF-Tu·GTP with the acceptor/T half of the tRNA (6)
appears to be applicable also to ternary complex formation by
EF-1
·GTP, as deduced by the interference role of the
phosphoribosyl modification in the T stem of initiator
tRNAMet and ternary complex formation by the tRNA
half-molecule 12 RNA. The binding discrimination against initiator tRNA
and uncharged tRNA, and tight ternary complex formation with various
charged tRNAs are also closely similar functions of EF-1
·GTP and
EF-Tu·GTP. These RNA-interaction properties are strongly conserved
between EF-Tu and EF-1
even though these proteins have only about
33% sequence identity. Notably, however, most of the sequence
variability between EF-Tu and EF-1
is away from the amino acids
contacting the aminoacyl-tRNA. These contact sites include many highly
conserved residues, particularly among those contacting the aminoacyl-
and 5'-termini, while much of the sequence variability is accommodated in loops (Fig. 4) distant from the RNA (6). This arrangement presents a
picture of a protein with a core structure that interacts with
aminoacyl-tRNA much like EF-Tu does, but with unique surface features
that presumably are involved in the cytoskeleton interaction (41) and
various roles in cellular regulation (42) that are characteristic of
higher eukaryotic EF-1
.