Direct Evidence of an Elongation Factor-Tu/Ts·GTP·Aminoacyl-tRNA Quaternary Complex

Burnett, Benjamin J.; Altman, Russ B.; Ferguson, A.D.; Wasserman, Mark; Zhou, Zhou; Blanchard, Scott C.

doi:10.1074/jbc.m114.583385

Cited by 20 publications

(55 citation statements)

References 59 publications

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“…This is in excellent agreement with kinetic studies in the bacterial system where GTP hydrolysis has been shown to be a very fast step, whereas tRNA accommodation and especially EF-Tu dissociation are rate-limiting during tRNA selection (Pape et al, 1998). Furthermore, our results rationalize recent studies on the formation and turnover of bacterial EF-Tu • GXP • EF-Ts • aa-tRNA quarternary complexes (Burnett et al, 2014; 2013), which proposed a novel role of EF-Ts in promoting release of aa-tRNA from EF-Tu • GDP. The presence of a second proofreading state containing A/T-tRNA, but lacking density for eEF1A, implies that also tRNA accommodation constitutes a second slow step after eEF1 A dissociation, in agreement with the hypothesis that necessary conformational changes in the tRNA elbow to allow A/T to A/A transition resemble a stochastic trial-and-error process and not a concerted pathway (Whitford et al, 2010; Geggier et al, 2010).…”

Section: Discussionsupporting

confidence: 89%

Structural Snapshots of Actively Translating Human Ribosomes

Behrmann

Loerke

Budkevich

et al. 2015

Cell

180

254

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Summary Macromolecular machines, such as the ribosome, undergo large-scale conformational changes during their functional cycles. While their mode of action is often compared to that of mechanical machines, a crucial difference is that at the molecular dimension, thermodynamic effects dominate functional cycles, with proteins fluctuating stochastically between functional states defined by energetic minima on an energy landscape. Here, we have used cryo-electron microscopy to image ex vivo-derived human polysomes as a source of actively translating ribosomes. Multiparticle refinement and three-dimensional variability analysis allowed us to visualize a variety of native translation intermediates. Significantly populated states include not only elongation cycle intermediates in pre- and post-translocational states, but also eEF1A-containing decoding and termination/recycling complexes. Focusing on the post-translocational state, we extended this assessment to the single-residue level, uncovering striking details of ribosome-ligand interactions and identifying both static and functionally important dynamic elements.

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Section: Discussionsupporting

confidence: 89%

Structural Snapshots of Actively Translating Human Ribosomes

Behrmann

Loerke

Budkevich

et al. 2015

Cell

180

254

View full text Add to dashboard Cite

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“…This model is supported by recent work from Blanchard and colleagues, who have shown that EF-Ts promotes both the association and dissociation of tRNA from EF-Tu (29). This activity is concomitant to yet distinct from the well-described GEF function of EF-Ts (30). Knudsen and colleagues recently suggested that the coiled-coil domain of EF-Ts, and in particular its conserved basic patch (including Arg219), could promote tRNA loading onto EF-Tu (31).…”

Section: Discussionsupporting

confidence: 58%

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

Jones

Garza-Sánchez

et al. 2017

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

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Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CT EC869 tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CT EC869 binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CT EC869 only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.cell communication | protein synthesis | toxin-immunity proteins | ribonuclease | bacterial competition

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“…By contrast, CdiA-CT NC101 activity is not detected in vitro unless both EF-Ts and EF-Tu are present. Despite this discrepancy, we propose that EF-Ts stimulates the activity of each toxin by increasing steady-state GTP·EF-Tu·aa-tRNA substrate levels as described by Blanchard and colleagues ( 68 , 69 ). Though EF-Tu has nanomolar affinity for aa-tRNA at 0–4°C ( 70 ), these interactions are estimated to be 20- to 30-fold weaker at 37°C ( 71 , 72 ), indicating that the dissociation constants approach the micromolar range under the conditions used here to monitor in vitro tRNase activity.…”

Section: Discussionmentioning

confidence: 56%

Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs

Michalska

Gucinski

Garza-Sánchez

et al. 2017

Nucleic Acids Research

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Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3′-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3′-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3′-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3′-end of aa-tRNA for entry into the nuclease active site.

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Direct Evidence of an Elongation Factor-Tu/Ts·GTP·Aminoacyl-tRNA Quaternary Complex

Cited by 20 publications

References 59 publications

Structural Snapshots of Actively Translating Human Ribosomes

Structural Snapshots of Actively Translating Human Ribosomes

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

Structure of a novel antibacterial toxin that exploits elongation factor Tu to cleave specific transfer RNAs

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