GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits

Holtkamp, Wolf; Cunha, C. E.; Peske, Frank; Konevega, Andrey L.; Wintermeyer, Wolfgang; Rodnina, Marina V.

doi:10.1002/embj.201387465

Cited by 92 publications

(189 citation statements)

References 87 publications

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“…Consistent with recently published reports (47,48), we found that the H92A mutation introduced into wild-type EF-G decreased the GTPase activity of EF-G by at least 100-fold (Fig. S8A).…”

Section: Ef-g Adopts Different Conformations In Pre-and Posttranslocasupporting

confidence: 92%

Following movement of domain IV of elongation factor G during ribosomal translocation

Salsi

Farah

Dann

et al. 2014

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

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Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G–ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.

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“…Consistent with recently published reports (47,48), we found that the H92A mutation introduced into wild-type EF-G decreased the GTPase activity of EF-G by at least 100-fold (Fig. S8A).…”

Section: Ef-g Adopts Different Conformations In Pre-and Posttranslocasupporting

confidence: 92%

Following movement of domain IV of elongation factor G during ribosomal translocation

Salsi

Farah

Dann

et al. 2014

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

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“…22. Our results suggest that EF-G serves as a force-generating motor via a power stroke in the early steps of translocation, supporting the proposal that energy released from GTP hydrolysis triggers EF-G conformational changes to promote translocation (24). The sequential motions of EF-G that follow the first stroke and complete…”

supporting

confidence: 79%

Elongation factor G initiates translocation through a power stroke

Chen

Cui

Beausang

et al. 2016

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

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During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

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“…In this assay system, we measure the complete translocation cycle time from the association of EF-G·GTP to the pre-translocation ribosome to the release of EF-G·GDP from the post-translocation ribosome. This approach contrasts those based on fluorescence-labeled mRNA, fluorescence-labeled tRNAs or those with puromycin reactivity as a marker for the post-termination ribosomal complex (see Holtkamp et al [28]). The method has been used to determine how the Mg 2+ ion concentration affects the rate of translocation at high and low concentration of EF-G, how the rate of translocation varies with the distance between the translocated codon and the initiation codon and how SD-like sequences within ORFs affect the translocation rate.…”

Section: Introductionmentioning

confidence: 96%

“…[28], Cunha et al[33] and Savelsbergh et al[35], these approaches estimate but fractions of the total translocation time and the mechanistic interpretation of the results is sometimes difficult. For instance, Cunha et alused one and the same mRNA labeled with two different Effect of the free Mg 2+ concentration on the translocation rate in the long ORF.…”

mentioning

confidence: 98%

Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

Borg

Ehrenberg

2015

Journal of Molecular Biology

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Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account.

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GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits

Cited by 92 publications

References 87 publications

Following movement of domain IV of elongation factor G during ribosomal translocation

Following movement of domain IV of elongation factor G during ribosomal translocation

Elongation factor G initiates translocation through a power stroke

Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

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