Quantitative Assessment of EF-1alpha ·GTP Binding to Aminoacyl-tRNAs, Aminoacyl-viral RNA, and tRNA Shows Close Correspondence to the RNA Binding Properties of EF-Tu*

Theo W. DreherDagger §, Olke C. Uhlenbeck, and Karen S. Browningparallel

From the Dagger  Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804, the  Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, and the parallel  Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1alpha ·GTP. Aminoacylated fully modified tRNAs and unmodified tRNA transcripts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) from higher plants or Escherichia coli were bound with Kd values between 0.8 and 10 nM. A valylated 3'-fragment of turnip yellow mosaic virus RNA, which has a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNAVal. Uncharged tRNA and initiator Met-tRNAMet from wheat germ, RNAs that are normally excluded from the ribosomal A site in vivo, bound weakly. The discrimination against wheat germ initiator Met-tRNAMet was almost entirely due to the 2'-phosphoribosyl modification at nucleotide G64, since removal resulted in tight binding by EF-1alpha ·GTP. A 44-nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro selection to bacterial EF-Tu formed an Ala-RNA·EF-1alpha ·GTP complex with a Kd of 29 nM, indicating that much of the binding affinity for aminoacylated tRNA is derived from interaction with the acceptor/T half of the molecule. The pattern of tRNA interaction observed for EF-1alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A).

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The translational elongation factors EF-1alpha (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-1alpha function to that of EF-Tu (see, e.g., Refs. 9-11) has been convincingly established by the interchangeability of EF-Tu and EF-1alpha in binding to, although not in supporting protein synthesis by, bacterial and mammalian ribosomes (12). EF-1alpha , 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-1alpha with tRNAs are almost non-existent. The only estimate for the stability of aminoacyl-tRNA·EF-1alpha ·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-1alpha ·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-1alpha ·GTP (14), although no dissociation constants were reported.

In order to improve our understanding of the detailed function of EF-1alpha in eukaryotic protein synthesis, we have studied the binding of wheat germ EF-1alpha ·GTP to various charged and uncharged RNAs. Our results emphasize the similarity in the RNA binding properties of higher eukaryotic EF-1alpha ·GTP to those of its bacterial homologue EF-Tu·GTP.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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-1alpha ·GTP Binding Assays-- Purified wheat germ EF-1alpha (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-1alpha ·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-1alpha ·GTP. Each binding assay was performed in duplicate, and repeated at least twice. The concentration of active EF-1alpha ·GTP present in binding experiments was determined by binding in the presence of excess [3H]Val-tRNAVal transcript. Aminoacyl-tRNA·EF-1alpha ·GTP ternary complex formation follows Equation 1.
K<SUB>d</SUB>=<FR><NU>[<UP>free aa-tRNA</UP>][<UP>free EF-1&agr;</UP> · <UP>GTP</UP>]</NU><DE>[<UP>aa-tRNA</UP> · <UP>EF-1&agr;</UP> · <UP>GTP</UP>]</DE></FR> (Eq. 1)
In terms of total concentrations of aminoacylated tRNA (t) and EF-1alpha ·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) ± radical ((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-1alpha ·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-1alpha ·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-1alpha ·GTP] = Kd (reporter), this latter term being determined in the binding assay lacking competitor.
K<SUB>d</SUB>(<UP>comp.</UP>)=<FR><NU>[<UP>free comp. RNA</UP>][<UP>free EF-1&agr;</UP> · <UP>GTP</UP>]</NU><DE>[<UP>comp. RNA</UP> · <UP>EF-1&agr;</UP> · <UP>GTP</UP>]</DE></FR> (Eq. 1a)
Thus, the above equation becomes Equation 2.
K<SUB>d</SUB>(<UP>comp.</UP>)=<FR><NU>{[<UP>total comp. RNA</UP>]−K<SUB>d</SUB> <UP>offset</UP>}K<SUB>d</SUB>(<UP>reporter</UP>)</NU><DE>K<SUB>d</SUB> <UP>offset</UP></DE></FR> (Eq. 2)

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 beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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-1alpha ·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-1alpha . 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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Table I
Dissociation constants for the binding of modified and unmodified aminoacyl-tRNAs by wheat germ EF-1alpha ·GTP

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-1alpha ·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-1alpha (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-1alpha 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-1alpha ·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-1alpha ·GTP to form tight complexes with RNAs of varying acceptor stem architecture. Tight complex formation was dependent on EF-1alpha activation and was not observed for EF-1alpha ·GDP, and valylated viral RNA competed with valylated tRNAVal for binding by EF-1alpha ·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-1alpha ·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").

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-1alpha ·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-1alpha ·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-1alpha ·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-1alpha ·GTP. At accessible concentrations of EF-1alpha ·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).

                              
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Table II
The interaction of wheat germ EF-1alpha ·GTP with elongator and initiator tRNAMet

Both the wheat germ and yeast initiator tRNAMet were oxidized with periodate in order to remove the 2'-ribosyl modification of nucleotide 64 by beta -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 beta -elimination procedure, permitting subsequent aminoacylation. Both oxidized initiator methionyl-tRNAs formed high affinity complexes with EF-1alpha , 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-1alpha ·GTP binding.

EF-1alpha ·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-1alpha ·GTP (Fig. 3). The Delta  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.

Alanyl-Delta Anticodon RNA was bound by wheat germ EF-1alpha ·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-1alpha ·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-1alpha ·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.

                              
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Table III
Interaction of EF-1alpha ·GTP with acceptor/T domain of RNAs derived from E. coli tRNAAla

Residues Contacting Aminoacyl-tRNA Are Conserved between EF-Tu and EF-1alpha -- The close similarity in RNA binding properties between bacterial EF-Tu and wheat germ EF-1alpha 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-1alpha .3 The sequence identity is scattered throughout the protein, permitting ready alignment of all EF-Tu and EF-1alpha 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-1alpha 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-1alpha s differ by some 7 insertions and 3 deletions from EF-Tu. Mammalian and plant EF-1alpha 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-1alpha . The EF-1alpha 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-1alpha 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.

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-1alpha 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-1alpha . Supportive experimental evidence comes from cross-linking studies between rabbit EF-1alpha ·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-1alpha . EF-1alpha has similar affinity for GTP and GDP, while EF-Tu has a 100-fold higher affinity for GDP than for GTP (13).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

High Affinity Binding of EF-1alpha ·GTP to Aminoacylated tRNAs-- The equilibrium dissociation constants of ternary complexes formed by wheat germ EF-1alpha ·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-1alpha ·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-1alpha ·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-1alpha are from different sources. The 12-fold range of Kd values we have observed for the aminoacyl-tRNA·EF-1alpha ·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-1alpha ·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-1alpha ·GTP (Kd values of 0.83 and 1.0 nM, respectively; Table I). The stabilities of ternary complexes formed with higher eukaryotic EF-1alpha ·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-1alpha ·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-1alpha ·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-1alpha ·GTP. Uncharged wheat germ tRNA interacted very weakly with EF-1alpha ·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-1alpha ·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-1alpha ·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-1alpha ·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-1alpha ·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-1alpha ·GTP Interacts Primarily with the Acceptor/T Half of Aminoacyl-tRNA-- The relatively tight complexes formed between EF-1alpha ·GTP and two alanylated deletion variants derived from tRNAAla (Delta  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-1alpha ·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-1alpha ·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-1alpha ·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-1alpha 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-1alpha ·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-1alpha ·GTP and EF-Tu·GTP. These RNA-interaction properties are strongly conserved between EF-Tu and EF-1alpha even though these proteins have only about 33% sequence identity. Notably, however, most of the sequence variability between EF-Tu and EF-1alpha 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-1alpha .

    ACKNOWLEDGEMENTS

We thank Jeff Pleiss for discussions, Linda Behlen for making the biotinylated oligonucleotides, Dr. K. Watanabe for advice on hybridization selection of tRNAs, Dr. W. Merrick for the gift of rabbit EF-1alpha , Dr. Andrew Feig for assistance with KaleidaGraph, and Drs. C. Florentz and R. Giegé for the gift of yeast tRNAMet.

    FOOTNOTES

* This work was supported by Scholar Grant SG-209 from the American Cancer Society (to T. W. D.); by National Institutes of Health Grants AI-33907, GM-54610 (to T. W. D.), and GM-37552 (to O. C. U.); and by National Science Foundation Grant MCB-9406601 (to K. S. B.). This is Technical Report 11303 from the Oregon Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 541-737-1795; Fax: 541-737-0497.

The abbreviation used is: TYMV, turnip yellow mosaic virus.

1 New names for EF-1alpha and EF-Tu (eEF1A and EF1A, respectively) are being adopted by IUPAC and IUBMB.

3 Information was obtained using the PredictProtein server (http://www.embl-heidelberg.de/predictprotein/ppDoPredDef.html).

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Top
Abstract
Introduction
Procedures
Results
Discussion
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