EF-G-Dependent GTPase on the Ribosome. Conformational Change and Fusidic Acid Inhibition

Seo, Hyuk‐Soo; Abedin, Sameem; Kamp, Detlev; Wilson, Daniel N.; Nierhaus, Knud H.; Cooperman, Barry S.

doi:10.1021/bi0516677

Cited by 73 publications

(104 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recent single-molecule investigations attempting to address this question suggest that EF-G can bind to the PRE complex with peptidyl-tRNA in both classical and hybrid configurations . Such findings are, indeed, consistent with earlier reports showing the rate of GTP hydrolysis is unaffected by tRNA positions within the PRE complex (Rodnina et al 1997;Katunin et al 2002;Seo et al 2006;Walker et al 2008). However, concrete conclusions regarding the conformation of the ribosome immediately prior to translocation were not obtained, in part due to the time resolution of the experiments performed.…”

Section: Discussionsupporting

confidence: 93%

Insights into the molecular determinants of EF-G catalyzed translocation

Wang¹,

Altman

Blanchard

2011

RNA

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 93%

Insights into the molecular determinants of EF-G catalyzed translocation

Wang¹,

Altman

Blanchard

2011

RNA

View full text Add to dashboard Cite

show abstract

“…FA appears to function as a slow inhibitor, permitting several rounds of GTP turnover by EF-G before complete inhibition is achieved (7,8), but it is not known whether the formation of FA·EF-G·GDP·ribosome complexes leads to shutdown of protein synthesis primarily by directly stalling the majority of ribosomes; by stalling a minority of ribosomes which then serve to blockade polysomes; or as a consequence of depleting the EF-G pool. The equilibrium of FAmediated inhibition will be affected by conditions that either drive the formation of FA·EF-G·GDP·ribosome complexes or favor their dissociation (e.g., high or low concentrations of FA, respectively) (8). Because FusB-type proteins facilitate the release of EF-G from the ribosome, and inhibit the formation of the FA-stalled complex, they will shift the equilibrium toward complex dissociation.…”

Section: Discussionmentioning

confidence: 99%

“…FA does not interfere with the primary catalytic function of EF-G, which involves the translocation of mRNA and associated tRNAs on the ribosome in a reaction coupled to the hydrolysis of GTP (7). Rather, FA acts to prevent dissociation of the resulting EF-G·GDP complex from the ribosome once translocation has occurred (5,8). Formation of ribosome·EF-G·GDP·FA complexes prevents further protein synthesis, with consequent cessation of bacterial growth.…”

mentioning

confidence: 99%

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Cox

Thompson

Jenkins

et al. 2012

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

View full text Add to dashboard Cite

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusBtype proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FAmediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome·EF-G·GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.antibacterial | protein synthesis | translocation | structure

show abstract

“…Subsequent recognition of the start codon allows P i release to occur, making hydrolysis of GTP irreversible. The mechanism of EF-G-dependent translocation is similar in that P i release occurs much more slowly than GTP hydrolysis (12,26) and is limited by a conformational rearrangement (12,40). Further experiments will be required to determine the internal equilibrium position of GTP and GDP⅐P i within ribosome-bound EF-G.…”

Section: Discussionmentioning

confidence: 99%

Role of hybrid tRNA-binding states in ribosomal translocation

Walker

Shoji

Pan

et al. 2008

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

Self Cite

108

View full text Add to dashboard Cite

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steadystate kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k trans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K 1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K 1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k trans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.elongation factor G ͉ translation ͉ exit site ͉ GTPase M ovement of tRNAs through the ribosome is believed to occur in a stepwise manner (1). Chemical protection experiments showed that, after peptidyl transfer, the acceptor ends of the tRNAs can move spontaneously with respect to the 50S subunit to form the hybrid state. In the hybrid state, the deacylated tRNA occupies the 30S P site and 50S E site (P/E site) while the peptidyl-tRNA occupies the 30S A site and 50S P site (A/P site). It was proposed that hybrid state formation precedes codon-anticodon movement within the 30S subunit, and only the latter event requires catalysis by elongation factor G (EF-G). Support for the hybrid-state model came from Förster resonance energy transfer (FRET) experiments in which an Ϸ20-Å movement of the 5Ј end of the newly deacylated tRNA toward ribosomal protein L1 was inferred after peptide bond formation, whereas the position of the peptidyl group changed little (2). Because L1 lies near the 50S E site, these data are consistent with movement of tRNA into the P/E site. Further evidence came from single-molecule FRET studies that monitored the distance between probes attached to the elbows of tRNAs in the A and P sites. Fluctuations between two distinct configurations were observed that were structurally consistent with the classical and hybrid states (3). More recently, a third state of the pretranslocation (PRE) complex was observed by single-molecule FRET, consistent with one tRNA bound to the P/E site and the other bound to the A/A site (4). Experiments that monitored mRNA in ribos...

show abstract

EF-G-Dependent GTPase on the Ribosome. Conformational Change and Fusidic Acid Inhibition

Cited by 73 publications

References 39 publications

Insights into the molecular determinants of EF-G catalyzed translocation

Insights into the molecular determinants of EF-G catalyzed translocation

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

Role of hybrid tRNA-binding states in ribosomal translocation

Contact Info

Product

Resources

About