Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu

Agirrezabala, Xabier; Frank, Joachim

doi:10.1017/s0033583509990060

Cited by 109 publications

(102 citation statements)

References 177 publications

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“…1; Samaha et al 1995;Kim and Green 1999). While the specific interactions of tRNAs in classical positions are critical for both peptide bond formation (Lieberman and Dahlberg 1994;Weinger et al 2004;Brunelle et al 2006;Dorner et al 2006) as well as peptide release (Feinberg and Joseph 2006), such contacts must be extensively remodeled for tRNA substrates to move directionally through the ribosome during active translation (Agirrezabala and Frank 2009;Munro et al 2009). …”

Section: Introductionmentioning

confidence: 99%

Insights into the molecular determinants of EF-G catalyzed translocation

Wang¹,

Altman

Blanchard

2011

RNA

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Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 39-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 39-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.

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Section: Introductionmentioning

confidence: 99%

Insights into the molecular determinants of EF-G catalyzed translocation

Wang¹,

Altman

Blanchard

2011

RNA

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“…Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1)(2)(3)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level.…”

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confidence: 99%

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

Adamczyk

Warshel

2011

Proc. Natl. Acad. Sci. U.S.A.

130

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The crucial process of aminoacyl-tRNA delivery to the ribosome is energized by the GTPase reaction of the elongation factor Tu (EF-Tu). Advances in the elucidation of the structure of the EF-Tu/ribosome complex provide the rare opportunity of gaining a detailed understanding of the activation process of this system. Here, we use quantitative simulation approaches and reproduce the energetics of the GTPase reaction of EF-Tu with and without the ribosome and with several key mutants. Our study provides a novel insight into the activation process. It is found that the critical H84 residue is not likely to behave as a general base but rather contributes to an allosteric effect, which includes a major transition state stabilization by the electrostatic effect of the P loop and other regions of the protein. Our findings have general relevance to GTPase activation, including the processes that control signal transduction.enzymatic catalysis | preorganization | allostery T he elongation cycle of protein synthesis uses the elongation factor Tu (EF-Tu) with GTP to deliver aminoacyl-tRNA (aa-tRNA) to the mRNA-programmed ribosome. More specifically, the GTP-bound state of EF-Tu forms a high-affinity ternary complex with the aa-tRNA. Upon binding of the ternary complex to the ribosome, the aa-tRNA occupies the A site and, when the codon-anticodon interaction is cognate, the GTPase activity of the EF-Tu is increased significantly. The conformational change following GTP hydrolysis to GDP and a leaving phosphate group (P i ) leads to dissociation of EF-Tu from the ribosome and accommodation of the aa-tRNA on the A site for peptidyl transfer (see Fig. 1 for a schematic description; for additional details, see refs. 1-3).Breakthrough in the elucidation of the ribosome structure (2-13) and careful biochemical studies (1-3, 14-25) allow one to begin thinking about the nature of the mechanism of the elongation process. In particular, the elucidation of structure of the EF-Tu/ribosome complex (our study refers to the structure of EF-Tu in the complex as EF-Tu′) (9) opens the way for the exploration of the activation process at the molecular level. However, despite major biochemical and structural breakthroughs, a detailed explanation of how the codon recognition in the 30S subunit leads to GTP hydrolysis remains elusive. That is, although it is known that the precise positioning of H84 is critical for efficient catalysis (1,9,(11)(12)(13)(23)(24)(25)(26), the energetics of this positioning and its ultimate role in stabilizing the transition state (TS) are unclear. More precisely, it is frequently assumed that the conserved H84 is moved to a catalytic configuration and then serves as a general base, but this assumption is problematic (see ref. 1 and Reproducing the Overall Catalytic and Mutational Effects below) and has similar pitfalls as in the highly related case of the Ras-RasGAP (RasGAP) system. That is, where it was originally assumed that Q61 in the RasGAP system serves as a general base, but this assumption has been shown to ...

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“…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)(2)(3)(4)(5)(6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7)(8)(9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10)(11)(12).…”

mentioning

confidence: 99%

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

Schrader

Chapman

Uhlenbeck

2011

Proc. Natl. Acad. Sci. U.S.A.

123

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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...

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Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu

Cited by 109 publications

References 177 publications

Insights into the molecular determinants of EF-G catalyzed translocation

Insights into the molecular determinants of EF-G catalyzed translocation

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

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

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