Control of phosphate release from elongation factor G by ribosomal protein L7/12

Savelsbergh, Andreas; Mohr, Dagmar; Kothe, Ute; Wintermeyer, Wolfgang; Rodnina, Marina V.

doi:10.1038/sj.emboj.7600884

Cited by 100 publications

(108 citation statements)

References 42 publications

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“…Our mant-GTP hydrolysis experiments, which monitor a conformational change in EF-G, suggest that the GЈ mutations perturb GTP hydrolysis and/or P i release from EF-G. These results are in partial accord with a study analyzing mutations in the CTD of L7/L12, which reported effects on P i release but surprisingly no significant effects on translocation (34).…”

Section: Discussionsupporting

confidence: 85%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking ␣-helix A G in the G subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix A G , caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix A G of EF-G and L7/L12 of the ribosome.

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Section: Discussionsupporting

confidence: 85%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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“…When different proteins compete for the same binding site, binding rate, not binding affinity, is the key determinant for which protein gets bound (5,8). The binding rate constants of elongation factors for the SRL-binding site on the ribosome are Ϸ10 8 M Ϫ1 s Ϫ1 at an ionic strength Ϸ100 mM (9,10), which is even somewhat higher than the binding rate constant of restrictocin at the same ionic strength (1). It can thus be suggested that ribotoxins need the high binding speed to compete against elongation factors effectively.…”

Section: Dissection Of the High Rate Constant For The Binding Of A Rimentioning

confidence: 99%

Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

Qin

Zhou

2009

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

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Restrictocin belongs to a family of site-specific ribonucleases that kill cells by inactivating the ribosome. The restrictocin–ribosome binding rate constant was observed to exceed 1010 M−1 s−1. We have developed a transient-complex theory to model the binding rates of protein–protein and protein–RNA complexes. The theory predicts the rate constant as ka = ka0 exp(−ΔGel*/kBT), where ka0 is the basal rate constant for reaching the transient complex, located at the outer boundary of the bound state, by random diffusion, and ΔGel* is the average electrostatic interaction free energy of the transient complex. Here, we applied the transient-complex theory to dissect the high restrictocin–ribosome binding rate constant. We found that the binding rate of restrictocin to the isolated sarcin/ricin loop is electrostatically enhanced by ≈300-fold, similar to results found in other protein–protein and protein–RNA complexes. The ribosome provides an additional 10,000-fold rate enhancement because of two synergistic mechanisms afforded by the distal regions of the ribosome. First, they provide additional electrostatic attraction with restrictocin. Second, they reposition the transient complex into a region where local electrostatic interactions of restrictocin with the sarcin/ricin loop are particularly favorable. Our calculations rationalize a host of experimental observations and identify a strategy for designing proteins that bind their targets with high speed.

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“…Therefore, we postulate that the hybrid-ratcheted configuration is the natural substrate for EF-G, and EF-G may either bind to that state preferentially or shift the ribosomes to that state before the tRNA movement on the 30S subunit. Translocation is then completed by coupling conformational changes of the factor with further ribosome rearrangements, resulting in tRNA movement and, presumably, the reverse rotation of the 30S subunit (19)(20)(21).…”

Section: Resultsmentioning

confidence: 99%

Structure of ratcheted ribosomes with tRNAs in hybrid states

Julián

Konevega

Scheres³

et al. 2008

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

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176

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During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G.translocation ͉ elongation factor G ͉ cryo-electron microscopy D uring the elongation cycle, the tRNAs⅐mRNA complex is translocated to allow reading of the following codon on mRNA. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis. During translocation, the ribosome changes from the pretranslocation state with deacylated tRNA in the peptidyl site (P site) and peptidyltRNA in the aminoacyl site (A site) to the posttranslocation state where A-and P-site tRNAs have moved to P and exit (E) sites, respectively. The universal design of ribosomes from two subunits inspired early suggestions that translocation may involve a movement of the subunits relative to each other (1, 2). Such models imply the existence of intermediate states for the tRNAs that differ from the classic A, P, and E states. Chemical probing experiments with pretranslocation ribosomes indicated that after peptidyl transfer the acceptor ends of the tRNAs spontaneously moved on the large 50S subunit toward their posttranslocation positions (3), indicating that peptidyl-tRNA and deacylated tRNA entered hybrid A/P and P/E states, respectively. The transition was observed in the absence of EF-G, and the driving force for the movement was attributed to different affinities of the A, P, and E sites on the 50S subunit (4) for the chemically different acceptor ends of the tRNAs (5, 6). Nevertheless, cryo-electron microscopy (cryoEM) of ribosomes in the pretranslocation state showed the tRNAs in their classic A, P, and E states (7). Although this result was difficult to reconcile with the hybrid-state model, it is important to note that in that complex the occupancy of the E site by deacylated tRNA may have prevented the P-site tRNA to enter the P/E-site hybrid state.CryoEM of ribosomes in complex with EF-G revealed a relative rotation between subunits referred to as ratc...

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Control of phosphate release from elongation factor G by ribosomal protein L7/12

Cited by 100 publications

References 42 publications

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

Structure of ratcheted ribosomes with tRNAs in hybrid states

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