Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Abstract

To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.T he ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1-6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7-9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10-12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein.It therefore seems unlikely that a minimum threshold binding af...

Two proofreading steps amplify the accuracy of genetic code translation

“…In the tRNA Phe case, the similar k dip values in the WT and W1 cases (Table S1) are much smaller than the maximal rate of peptide bond formation (26) and must therefore reflect similar k cat /K m values for the reaction. We suggest that the significantly smaller k dip values for the "strong" T1 and T2 variants in relation to the WT and W1 variants reflect slow release of EF-Tu·GDP from the ribosome, as previously proposed (23). In the tRNA Glu case, the k dip value is similar among the WT, T1, and T2 variants, reflecting similar k cat /K m values for peptide bond formation.…”

“…We found that proofreading amplification of the accuracy of codon reading by aa-tRNAs, F, increases linearly with the affinity (K A value) of aa-tRNAs to EF-Tu·GTP (Fig. 5) when K A is varied by T-stem mutations (23). From this finding, we propose that EF-Tu plays a fundamental role not only in initial codon selection by ternary complex in the GTP conformation but also in the rechecking of the initial codon choice in a proofreading step following GTP hydrolysis on EF-Tu, Pi release, and conformational change of the factor (Fig.…”

“…2F), such a low Mg 2+ sensitivity could be typical of a second proofreading step after dissociation of EF-Tu·GDP from the ribosome. We decided to subject this hypothesis to stringent testing in a series of experiments that were inspired by the observation that the choice of base pairs in the T stem of any tRNA strongly affects its affinity to EF-Tu · GTP (23). For these experiments, we first developed a kinetic model for two-step proofreading in ribosomal protein synthesis in bacteria (Fig.…”

Histidine 66 in Escherichia coli Elongation Factor Tu Selectively Stabilizes Aminoacyl-tRNAs

“…The equilibrium dissociation constant (K D ) was measured in buffer A as described in Sanderson and Uhlenbeck (20). The apparent rate of GTP hydrolysis was performed in buffer B (50 mM Hepes (pH 7.0), 30 mM KCl, 70 mM NH 4 Cl, 10 mM MgCl 2 , and 1 mM DTT) with 1 M ribosomes as described in Schrader et al (22). The rate of dipeptide formation (k pep ) was performed in buffer B as described in Schrader et al (22).…”

“…The apparent rate of GTP hydrolysis was performed in buffer B (50 mM Hepes (pH 7.0), 30 mM KCl, 70 mM NH 4 Cl, 10 mM MgCl 2 , and 1 mM DTT) with 1 M ribosomes as described in Schrader et al (22). The rate of dipeptide formation (k pep ) was performed in buffer B as described in Schrader et al (22). The ternary complex binding assay was performed in buffer B as described in Ledoux and Uhlenbeck (23).…”