A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA^Ala-based body (tRNA^AlaB) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA^PheB body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA^AlaB body than from the tRNA^PheB body. At ∼1 µM EF-Tu, tRNA^AlaB conferred considerably faster incorporation kinetics than tRNA^PheB, especially in the case of the bulky bK. In contrast, the swap to the tRNA^AlaB body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA^AlaB and tRNA^PheB bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.     

Histidine 66 in Escherichia coli elongation factor tu selectively stabilizes aminoacyl-tRNAs

The universally conserved His-66 of elongation factor Tu (EF-Tu) stacks on the side chain of the esterified Phe of Phe-tRNA(Phe). The affinities of eight aminoacyl-tRNAs were differentially destabilized by the introduction of the H66A mutation into Escherichia coli EF-Tu, whereas Ala-tRNA(Ala) and Gly-tRNA(Gly) were unaffected. The H66F and H66W proteins each show a different pattern of binding of 10 different aminoacyl-tRNAs, clearly showing that this position is critical in establishing the specificity of EF-Tu for different esterified amino acids. However, the H66A mutation does not greatly affect the ability of the ternary complex to bind ribosomes, hydrolyze GTP, or form dipeptide, suggesting that this residue does not directly participate in ribosomal decoding. Selective mutation of His-66 may improve the ability of certain unnatural amino acids to be incorporated by the ribosome.     

Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

In most prokaryotes Asn-tRNA^Asn and Gln-tRNA^Gln are formed by amidation of aspartate and glutamate mischarged onto tRNA^Asn and tRNA^Gln, respectively. Coexistence in the organism of mischarged Asp-tRNA^Asn and Glu-tRNA^Gln and the homologous Asn-tRNA^Asn and Gln-tRNA^Gln does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNA^Asn intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNA^Asn from Asn-tRNA^Asn and Asp-tRNA^Asp by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs.     

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.     

Influence of the last amino acid in the nascent peptide on EF-Tu during decoding

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249–257; Björnsson et al (1996) EMBO J 15, 1696–1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA^Trp by varying the codons at positions −1 and −2 at the 5′ side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J 4, 1049–1052). The effect on readthrough by changing these −1 and −2 codons is different on the two forms of tRNA^Trp and is also dependent on the structure of EF-Tu. Readthrough by the tRNA^Trp-derived suppressor, but not wild-type tRNA^Trp, is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA^Trp are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA^Trp at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509–1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756–3760) were found not to be altered in sensitivity to the last two amino acids in the nascent peptide.     

Changeability of individual domains of an aminoacyl-tRNA in polymerization by the ribosome

The changeabilities of individual modules of aminoacyl-tRNAs are poorly understood, despite the relevance for evolution, translational accuracy and incorporation of unnatural amino acids (AAs). Here, we dissect the effect of successive changes in four domains of on translation in a purified system. Incorporating five AAs, not one, was necessary to reveal major effects on yields of peptide products. Omitting tRNA modifications had little affect, but anticodon mutations were very inhibitory. Surprisingly, changing the terminal CCA to CdCA was sometimes inhibitory and non-cognate AAs were sometimes compensatory. Results have implications for translational fidelity and engineering.     

Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3′-OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is effectively determined only by the chemical nature of the amino acid side chain. Even the chirality of the side chain exhibits little influence. Proline confers the lowest stability to the linkage, while isoleucine and valine confer the highest, whereas the nucleotide sequence in the tRNA provides negligible contribution to the stability. We find that, among the variables tested, the protein translation factor EF-Tu is the only one that can protect a weak acyl linkage from hydrolysis. These results suggest that each amino acid plays an active role in determining its own stability in the acyl linkage to tRNA, but that EF-Tu overrides this individuality and protects the acyl linkage stability for protein synthesis on the ribosome.     

Translational regulation by modifications of the elongation factor Tu

EF-Tu fromE. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorlation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys⁵⁶ which is converted to Me₂Lys upon entering the stationary phase. Lys⁵⁶ is in the GTPase switch-1 regions (residues 49–62), a strongly conserved site involved in interactions with the nucleotide and the 5′ end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy.In vivo 5–10% of EF-Tu is phosphorylated at Thr³⁸² by a ribosome-associated kinase. In EF-Tu-GTP, Thr³⁸² in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu¹¹⁷ that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyl tRNA. Thein vivo role of this reversibles modification is still a matter of speculation. T4 infection ofE. coli may trigger a phage-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly⁵⁹-Ile⁶⁰ in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanisms ofin cis inhibition of late T4 mRNA translation. Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe

The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution.     

The Role of Initiator tRNAi met in Fidelity of Initiation of Protein Synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA_i ^met compared to the one for the elongator tRNAs indicates that only the initiator tRNA_i ^met specifies the amino acid side chain.     

Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.     

The site of interaction of aminoacyl-tRNA with elongation factor Tu

We have used RNases T1, T2 and A to digest two aminoacyl-tRNAs, Escherichia coli Phe-tRNAPhe and E. coli Met- tRNAMetm both in the naked forms and in ternary complexes with E. coli elongation factor Tu (EF-Tu) and GTP. An analysis of the 'footprinting' results has led to an interpretation that has localized the part of the three-dimensional structure of aminoacyl-tRNA covered by the protein in the ternary complex. In terms of the three-dimensional structure of tRNA established for yeast tRNAPhe, EF-Tu covers the aa-end, aa-stem, T-stem, and extra loop on the side of the L-shaped tRNA that exposes the extra loop.     

Assessing post-anthesis nitrogen uptake, distribution and utilisation in grain protein synthesis in barley (Hordeum vulgare L.) using 15N fertiliser and 15N proteinogenic and non-proteinogenic amino acids

We investigated the effects of nitrogen (N) availability during the vegetative phase on (a) post‐anthesis N uptake and (b) its translocation into ears in barley plants grown in a greenhouse at two levels of N: low (50 mg N kg^?1 sand) and optimal N supply (150 mg N kg^?1 sand). Plants in the two N treatments were fertilised with the same amount of labelled ¹⁵N [50 mg ¹⁵N kg^?1 sand at 10% ¹⁵N_exc (N_excess, i.e. N_exc, is defined as the abundance of enriched stable isotope minus the natural abundance of the isotope) applied as ¹⁵NH₄¹⁵NO₃] 10 days after anthesis (daa). In a separate experiment, the uptake and transport into ears of proteinogenic and non‐proteinogenic amino acids were studied to determine whether a relationship exists between amino acid transport into ears and their proteinogenic nature. Plants were fed with either ¹⁵N‐α‐alanine, a proteinogenic amino acid, or ¹⁵N‐α‐aminoisobutyric acid, a non‐proteinogenic amino acid. Both these amino acids were labelled at 95.6% ¹⁵N_exc. Results showed that N accumulations in stems, leaves and especially in ears were correlated with their dry matter (dm) weights. The application of 150 mg N kg^?1 sand significantly increased plant dm weight and total N accumulation in plants. During their filling period, ears absorbed N from both external (growth substrate) and internal (stored N in plants) sources. Nitrogen concentration in ears was higher in optimal N‐fed plants than in low N‐fed plants until 10 daa, but from 21 to 35 daa, differences were not detected. Conversely, ¹⁵N_exc in ears, leaves and stems was higher in low N‐fed plants than in optimal N‐fed plants. Ears acted as strong sink organ for the post‐anthesis N taken up from the soil independently of pre‐anthesis N nutrition: on average, 87% of the N taken up from the soil after anthesis was translocated and accumulated in ears. Low N‐fed plants continued to take up N from the post‐anthesis N fertiliser during the later grain‐filling period. The increase of pre‐anthesis N supply rate led to a decrease in the contribution of nitrogen derived from post‐anthesis ¹⁵N‐labelled fertiliser (N_dff) to total N in all aboveground organs, especially in ears where 44% and 22% of total N originated from post‐anthesis N uptake in low N‐fed and optimal N‐fed plants, respectively. The experiment with labelled amino acids showed that there was greater transport of proteinogenic amino acid into the ear (50% of total ¹⁵N) than non‐proteinogenic amino acid (39%). However, this transport of the non‐proteinogenic amino acids into ear suggested that the transport of N compounds from source (leaves) to sink organs (ear) might not be intrinsically regulated by their ability to be incorporated into storage protein of ears.     

Biosynthesis of cyclosporin A

Short term feeding of the mould Tolypocladium inflatum with ¹⁴C-labelled amino acids revealed a selective incorporation of l-leucine, l-valine, glycine and d, l-alanine into cyclosporins A and C. Feeding of l-[Me-¹⁴C]methionine exclusively labelled the N-methyl moieties of the cyclosporins. The distribution of radioactivity from this substrate was directly proportional to the number of the relevant N-methyl amino acids in cyclosporin A, indicating a simultaneous methylation of these residues.     

The 51-63 base pair of tRNA confers specificity for binding by EF-Tu

Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.     

Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

Amino acid chalcogen analogues as tools in peptide and protein research

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur‐containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl‐tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl‐ and phenylalanyl‐tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl‐tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.     

Is the sequence-specific binding of aminoacyl-tRNAs by EF-Tu universal among bacteria?

Three base pairs in the T-stem are primarily responsible for the sequence-specific interaction of tRNA with Escherichia coli and Thermus thermophilus EF-Tu. While the amino acids on the surface of EF-Tu that contact aminoacyl-tRNA (aa-tRNA) are highly conserved among bacteria, the T-stem sequences of individual tRNA are variable, making it unclear whether or not this protein-nucleic acid interaction is also sequence specific in other bacteria. We propose and validate a thermodynamic model that predicts the ΔG° of any tRNA to EF-Tu using the sequence of its three T-stem base pairs. Despite dramatic differences in T-stem sequences, the predicted ΔG° values for the majority of tRNA classes are similar in all bacteria and closely match the ΔG° values determined for E. coli tRNAs. Each individual tRNA class has evolved to have a characteristic ΔG° value to EF-Tu, but different T-stem sequences are used to achieve this ΔG° value in different bacteria. Thus, the compensatory relationship between the affinity of the tRNA body and the affinity of the esterified amino acid is universal among bacteria. Additionally, we predict and validate a small number of aa-tRNAs that bind more weakly to EF-Tu than expected and thus are candidates for acting as activated amino acid donors in processes outside of translation.     

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum

When the K⁺ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.     

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes was obtained using a functional analogue of aminoacyl-tRNA. Since elongation factor Tu (EF-Tu) screens both the modified aminoacyl-tRNAs and the ribosomal complexes for active particles, only functional macromolecular complexes are examined. This approach also provides an unequivocal identification of the transfer RNA binding site from which affinity labeling occurs. N^ε-bromoacetyl-Lys-tRNA was prepared by covalently attaching an electrophilic group to the side-chain of the amino acid. This chemical modification did not interfere with function, since the ?BrAcLys-tRNA participated successfully in EF-Tu and poly(rA)-dependent binding to ribosomes, peptide bond formation, and elongation factor G (EF-G)-mediated translocation. Affinity labeling of ribosomal RNA was observed only in those incubations which contained both EF-Tu and EF-G. The crosslinking of ?BrAcLys-tRNA to 23 S rRNA was found even if fusidic acid was added to the incubation before EF-G. The dependence of the covalent reaction on EF-G demonstrates, unambiguously, that a reactive residue of 23 S rRNA is located adjacent to the 3′ end of the functionally defined P site. Similarly, the affinity labeling of proteins L13/14/15, L2, L32/33, and L24 required EF-G-dependent translocation of ?BrAcLys-tRNA into the P site. Protein L27 was alkylated following the EF-Tu-dependent binding of ?BrAcLys-tRNA to the ribosome, and the extent of affinity labeling was stimulated by the addition of EF-G to the incubation. Double-label dipeptide experiments confirmed that affinity labeling occurred from functional tRNA binding sites by demonstrating that the same ?BrAcLys-tRNA which reacted covalently with 23 S rRNA or a ribosomal protein could also participate in peptide bond formation. Finally, the ribosome affinity labeling obtained with ?BrAcLys-tRNA · EF-Tu · guanylylimidodiphosphate differed little from that obtained with ?BrAcLys-tRNA · EF-Tu · GTP. This work constitutes the first direct examination of the aminoacyl ends of the EF-Tu-dependent conformational states of the ribosomal complex, and demonstrates the potential value of functional Lys-tRNA analogues with different probes attached to the lysine side-chain.