The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold

The refined crystal structure of the ternary complex of yeast Phe-tRNA^Phe, Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, revelas many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNA^Sec) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3′-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.     

Isolation, characterization and structural implications of a nuclease-digested complex of aminoacyl transfer RNA and Escherichia coli elongation factor Tu.

The complex of Escherichia coli elongation factor Tu with yeast Phe-tRNA^Phe was digested with T₁ ribonuclease. From the reaction mixture, a partially digested Phe-tRNA^Phe firmly bound to Tu was isolated. Analysis of the partially digested, tightly bound Phe-tRNA^Phe shows it has cleavages in the dihydrouridine and T ΨC loops. This suggests a non-essential role for these two loops in the binding of aminoacyl-tRNA to Tu. Also, since interactions between these loops are an important part of the system of tertiary interactions in tRNA, the results imply that these tertiary structural features are not essential for the binding. In separate experiments, direct shielding from nuclease attack of the 3′-terminus of the bound tRNA was also demonstrated. Based on these results, and those of other investigators, it is proposed that Tu binds primarily along the amino acid acceptor-T ΨC helix, and avoids contact with the various tRNA loops.     

The Effect of Modification of Nucleotide-37 on the Interaction of Aminoacyl-tRNA with the A Site of the 70S Ribosome

To estimate the effect of modified nucleotide 37, the interaction of two yeast aminoacyl-tRNAs (Phe-tRNA^Phe _+Y and Phe-tRNA^Phe _–Y) with the A site of complex [70S · poly(U) · deacylated tRNA^Phe in the P site] was assayed at 0–20°C. As comparisons with native Phe-tRNA^Phe _+Y showed, removal of the Y base decreased the association constant of Phe-tRNA^Phe _–Y and the complex by an order of magnitude at every temperature tested, and increased the enthalpy of their interaction by 23 kJ/mol. When the Y base was present in the anticodon loop of deacylated tRNA^Phe bound to the P site of the 70S ribosome, twice higher affinity for the A site was observed for Phe-tRNA^Phe _–Y but not for Phe-tRNA^Phe _+Y. Thus, the modified nucleotide 3" of the Phe-tRNA^Phe anticodon stabilized the codon–anticodon interaction both in the A and P sites of the 70S ribosome.     

A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

The hydrolysis of several aminoacylated transfer RNAs, by double-strand-specific ribonuclease from Naja oxiana was studied. The sensitivity to this enzyme of Phe-tRNA^Phe, Glu-tRNA^Glu and Met-tRNA_m^Met from Escherichia coli and Phe-tRNA^Phe from yeast was examined, both in the free state and complexed to E. coli elongation factor Tu. The hydrolysis patterns in the isolated state were similar for all aminoacylated tRNAs except Glu-tRNA₂^Glu, which exhibited striking differences probably arising from the existence of several subpopulations of tRNA₂^Glu. When engaged in a ternary complex with EF-Tu and GTP, the aminoacyl-tRNAs were efficiently protected in the amino acid acceptor and TΨC helices, showing that the interaction with EF-Tu primarily takes place at the -C-C-A end and at the amino acid acceptor and TΨC helices. In all cases an increased reactivity of the anticodon stem was observed in the complexed tRNA, possibly resulting from a conformational change in this region of the tRNAs.     

Discrimination between aminoacyl groups on su+ 7 tRNA by elongation factor Tu

The su⁺7 nonsense suppressor of Escherichia coli is a mutant tRNA^Trp that can be aminoacylated with either tryptophan or glutamine. We have compared the ternary complexes of glutaminyl and tryptophanyl-su⁺7 tRNA with elongation factor Tu and GTP. Glutaminyl-su⁺7 tRNA binds more strongly than tryptophanyl-su⁺7 tRNA to EF Tu · GTP. The greatest distinction between the two species of the tRNA is seen in their dissociation rates from the complex, which differ by as much as fivefold. The distinction is affected by pH values around neutrality. These results show that EF Tu can distinguish between two aminoacyl-tRNAs which differ only in the aminoacyl group. The implications for the unusual amino acid specificity of su⁺7 tRNA are discussed.     

The Solution Structure of Yeast tRNAPhe as Studied by Nuclear Overhauser Effects in NMR

Abstract

Recently, the imino proton spectrum of yeast tRNA^Phe has been assigned by means of the application of the nuclear Overhauser effect (NOE). In the present paper it will be shown that even for tRNA (MW 28000) connectivities between the imino proton spins can be observed using two-dimensional NOE spectroscopy. In this way the imino proton resonances of the D-stem region are assigned. The results are discussed in relation to those obtained by the classical one-dimensional nuclear Overhauser effect. It turns out that in 2D-NOE experiments connectivities from overlapping resonances can be observed which cannot be determined by one-dimensional Overhauser experiments. Moreover, the total assignment of the imino proton spectrum of yeast tRNA^Phe is used to relate the three-dimensional crystal structure of the tRNA to its solution structure. It is shown that the principle elements of the X-ray structure, i.e. the hydrogen bonding network and the stacking of the stems upon one another, are also found in solution. This is true for the presence as well as for the absence of magnesium ions. However, in absence of magnesium ions the tRNA structure appears to differ in details from that in the presence of magnesium ions. Finally, the influence of the elongation factor Tu from B.stearothermophilus on the tRNA structure is discussed.     

Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu

The interaction of three different Met-tRNAsMet from E. coli with bacterial elongation factor (EF) Tu X GTP was investigated by affinity chromatography. Met-tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF-Tu X GTP, while Met-tRNAmMet has a high affinity for the elongation factor. A modified Met-tRNAfMet which has a C1-G72 base pair binds much more strongly to immobilized EF-Tu X GTP than the native aminoacyl(aa)-tRNA with non-base-paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa-tRNA X EF-Tu X GTP ternary complex formation.     

Letters to the editor: Nuclear magnetic resonance studies of protein-RNA interactions. I. The elongation factor Tu-GTP aminoacyl-tRNA complex

The 300 MHz high-resolution nuclear magnetic resonance spectra of hydrogen-bonded protons in Escherichia coli tRNA^Glu and yeast tRNA^Phe have previously been reported and the resolved resonances assigned to specific base-pairs. Here we show that in complexes of these two tRNAs with elongation factor Tu there is no discernible loss of base-paired protons. Within the experimental accuracy this means that no helical arms open upon complex formation.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

The ribosome‐bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet‐tRNA^fMet to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet‐tRNA^fMet to the ribosome in a ternary complex, IF2·GTP·fMet‐tRNA^fMet. By using rapid kinetic techniques, we show here that binding of IF2·GTP to the 30S ribosomal subunit precedes and is independent of fMet‐tRNA^fMet binding. The ternary complex formed in solution by IF2·GTP and fMet‐tRNA is unstable and dissociates before IF2·GTP and, subsequently, fMet‐tRNA^fMet bind to the 30S subunit. Ribosome‐bound IF2 might accelerate the recruitment of fMet‐tRNA^fMet to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF‐Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.     

Ribosomal proteins and elongation factors

Structural work on the translation machinery has recently undergone rapid progress. It is now known that six out of nine ribosomal proteins have an RNA-binding fold, and two domains of elongation factors Tu and G have very similar folds. In addition, the complex of EF-Tu with a GTP analogue and Phe-tRNA^Phe has a structure that overlaps exceedingly well with that of EF-G·GDP. These findings obviously have functional implications.     

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt''s), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA^Phe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA^His and Phe-tRNA^Phe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt''s and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt''s.     

Assignment of the low field proton nuclear magnetic resonance spectrum of yeast phenylalanine transfer RNA to specific base pairs

High-resolution proton nuclear magnetic resonance spectra at 220 and 300 MHz have been used to investigate the base-pairing structure of fragments of yeast tRNA^Phe, of chemically modified tRNA^Phe and of intact tRNA^Phe. To a very good approximation the positions of the fragment spectra are additive within 0·2 part per million, indicating that factors responsible for certain structural features in the intact molecule are already present in the smaller fragments (half molecules, hairpins and $\text{3}\text{4}$ molecules). A simple first-order ring-current shift theory taken in conjunction with the cloverleaf model for tRNA^Phe (RajBhandary et al., 1967) has been used to predict the low-field (? 15 to ?11 part per million) nuclear magnetic resonance spectra and make assignments of the resolved resonances to ring NH protons of specific base pairs. The general agreement between the predicted and observed spectra to within 0·2 part per million confirms in detail the cloverleaf model for the secondary structure of tRNA^Phe in solution. It is also established that ring-current shifts are the principal factor responsible for the wide range of shifts observed in the low-field spectra. As a result it is evident that the resonances are very sensitive to small changes in the secondary structure and in some cases changes in the interbase distance as small as 0·2 Å could easily be detected. It is also clear from the analysis that certain of the resonances are sensitive to the tertiary structure of the molecule and specific examples are discussed. As with our previous study, we find no evidence for any strong Watson-Crick type base pairs beyond those predicted by the cloverleaf structure.     

Aminoacyl RNA domain of turnip yellow mosaic virus Val-RNA interacting with elongation factor Tu

Turnip yellow mosaic virus (TYMV) Val-RNA forms a complex with the peptide elongation factor Tu (EF-Tu) in the presence of GTP: the Val-RNA is protected by EF-Tu·GTP from non-enzymatic deacylation and nuclease digestion. The determination of the length of the shortest TYMV Val-RNA fragment that binds EF-Tu·GTP leads us to conclude that the valylated aminoacyl RNA domain equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T arm is sufficient for complex formation. Since the aminoacyl RNA domain is also sufficient for adenylation by the ATP(CTP):tRNA nucleotidyltransferase, an analogy can be drawn between these two tRNA-specific proteins.     

Domains of phenylalanyl-tRNA synthetase from Thermus thermophilus required for aminoacylation

The contribution of entire domains or particular amino acid residues of the phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus to the interaction with tRNA^Phe was studied. Removal of domain 8 of the β subunit resulted in drastic reduction of the dissociation constant of the FRS·tRNA^Phe complex. Neither the removal of arginine 2 of the β subunit, which makes the only major contact between domains β1–5 and the tRNA, nor the replacement of the conserved proline 473 by glycine had an influence on the aminoacylation activity of the FRS. Thus, the body comprising domains 1–5 of the β subunit may not be essential for efficient aminoacylation of tRNA^Phe by the FRS and rather be involved in other functions.     

Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in amino-acyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.EF-Tu.GTP ternary complex formation have been examined spectroscopically. Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val). Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-8. Upon association with EF-Tu.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.GTP. Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu-GTP. Thus, the binding of EF-Tu.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species. It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome. Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNA(Phe), the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group. The Kd of the tRNA(Phe).EF-Tu.GTP ternary complex was determined, at equilibrium, to be 2.6 microM by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein. Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6 degrees C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of elongation factor Tu from Escherichia coli with respect to attachment to the amino acid to the 2' or 3'-hydroxyl group of the terminal adenosine of tRNA.

Modified Tyr-tRNATyr and Phe-tRNAPhe species from yeast having the aminoacyl residue bound specifically to the 2' and 3' position of the terminal adenosine, respectively, were investigated for their ability to form ternary complexes with Escherichia coli elongation factor Tu and GTP. Both Tyr-tRNATyr-CpCpA (2'd) and Tyr-tRNATyr-CpCpA(3' d) derivatives which are esterified with the amino acid on the 3' and 2' position respectively and which lack the vicinal hydroxyl were able to form ternary complexes. The stability of these ternary complexes was lower than in the case of native Tyr-tRNATyr-CpCpA. Tyr-tRNATyr-CpCpA(3' d) having the amino acid attached to the 2' position interacted considerably more strongly with EF-Tu - GTP than Tyr-tRNATyr-CpCpA(2' d). Ternary complex formation was observed with neither Phe-tRNAPhe-CpCpA(2'NH2) nor Phe-tRNAPhe-CpCpA(3'NH2). It is concluded that 2' as well as 3' isomers of native aminoacyl-tRNA can be utilized for ternary complex formation but in a following step a uniform 2'-aminoacyl-tRNA - EF-Tu - GTP complex is formed. Although the free vicinal hydroxyl group of the terminal adenosine is not absolutely required, replacement of the ester linkage through with the amino acid is attached to tRNA by an amide linkage leads to loss of ability to interact with elongation factor Tu.     

Conformational changes of aminoacyl-tRNA and uncharged tRNA upon complex formation with polypeptide chain elongation factor Tu

The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP.     

High resolution NMR study of the melting of yeast tRNA Phe

The 300 MHz NMR spectra of the hydrogen bonded NH ring protons of tRNA_Yeast^Phe have been measured as a function of temperature. In the presence of Mg⁺⁺ two resonances, one from the Aψ base pair and the other probably from the neighboring base pair, disappear between 56 and 58°C. In the absence of Mg⁺⁺ the DHU stem, the acceptor stem (in particular its AU base pair #6 and #7) and the Aψ base pair in the anticodon stem melt slightly earlier than the other parts of the molecule. Since the DHU stems in tRNA_Yeast^Phe and tRNA_Coli^fMet have the same base pairing scheme it is interesting that their melting behavior is entirely different in both molecules. This is discussed in terms of the tertiary structure.     

Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA

The ribonuclease resistance assay has been used to probe the effect of trypsin modification of the Escherichia coli elongation factor Tu X GTP on the interaction with E. coli aminoacyl-tRNAs. First, the equilibrium dissociation constant of the trypsin-modified Tu X GTP X Thr-tRNA complex was determined to be 2.3 (0.1) X 10(-5)M at 4 degrees C, pH 7.4. Second, binding of 17 of 20 noninitiator aminoacyl-tRNAs and four sets of purified isoacceptor tRNAs to the modified protein was measured. At 4 degrees C, the complex stabilities vary 500-fold over the range of aminoacyl-tRNAs, with Gln-tRNA forming the strongest ternary complex and Val-tRNA, the weakest. The results are compared to a similar study of ternary complex formation using intact elongation factor Tu X GTP, and the major differences are discussed. An analysis of both data sets, particularly that for the leucine isoacceptor tRNAs, suggests that the trypsin modification of elongation factor Tu X GTP disrupts a region of protein that is involved with the aminoacyl side chain rather than that of the acceptor stem helix region of the aminoacyl-tRNA.