Thiostrepton inhibition of tRNA delivery to the ribosome

Gonzalez, Ruben L.; Chu, Steven; Puglisi, Joseph D.

doi:10.1261/rna.499407

Cited by 48 publications

(62 citation statements)

References 28 publications

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“…Thiostrepton is known to interfere with the ribosome function by binding to GAC (24). The lifetime of inhibited GTPase-activated state attributable to the presence of thiostrepton is found to be Ͻ100 ms, and essentially no Phe-tRNA Phe achieves the stabilized mid-FRET state or full accommodation (25). These observations suggest that the shorter lifetime component of mid-FRET state (or pseudo-GTPase-activated state) represents a state where the ternary complex forms a partial contact with SRL only, instead of both SRL and GAC.…”

Section: Discussionmentioning

confidence: 98%

The role of fluctuations in tRNA selection by the ribosome

Lee

Blanchard

Kim

et al. 2007

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

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144

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The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is ''induced fit'' between the ribosome and tRNA. By using singlemolecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.ribosome dynamics ͉ ribosome selection ͉ single-molecule FRET T ranslation, protein synthesis by the ribosome, is a vital cellular process involving the two-subunit ribosomal particle, multiple RNAs, and protein cofactors (1, 2). To synthesize a protein molecule with correct sequence, the ribosome has to select correct transfer RNA (tRNA) as dictated by messenger RNA (mRNA). In the decoding site of the ribosome, the proper matching of the 3-nt codon sequence of the mRNA to the anticodon sequence of tRNA occurs at a rate of 20-30 per second with an error rate of 10 Ϫ4 (3).The difference in base-pairing energy between codon and anticodon cannot explain the low error rate of translation (4). In the past decades, considerable progress has been made in understanding the ingenious mechanisms used by the ribosome to achieve this low error rate (2, 4-6). The overall selection process consists of initial selection of the ternary complex [aminoacyl-tRNA (aa-tRNA), GTP, and elongation factor Tu (EF-Tu)], GTP hydrolysis in EF-Tu, and then proofreading (6). The GTP hydrolysis ensures that initial selection is separated from the proofreading by an irreversible reaction. This two-step selection yields overall probability of an error to be the product of the error probabilities of the two selection steps, thus dramatically lowering the overall chance of error (7).The initial selection of aa-tRNA consists of initial binding of the ternary complex on the ribosome, interaction between tRNA and mRNA (the codon recognition), and stabilization of the ternary complex onto the ribosome. GTPase activation takes place after additional stabilizing contacts are formed between the ribosome and the ternary complex (6,8). In the initial selection of aa-tRNA, the ternary complex binding induces structural changes in the ribosome (''induced-fit'') (5, 9) in which the 30S subunit of the ribosome changes its structure and forms additional stabilizing contacts with the incoming tRNA after the codon recognition (9). In case of one base-mismatch out of the 3 bp (near cognate), additional binding contacts are weaker, presumably because the mismatched bases form a less compact structure for the ribosome to ''wrap'' around as it forms additional induced fit contacts. In other words, the tighter binding of cognate tRNA relative to near-cognate tRNA mainly is attributable...

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Section: Discussionmentioning

confidence: 98%

The role of fluctuations in tRNA selection by the ribosome

Lee

Blanchard

Kim

et al. 2007

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

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144

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“…All smFRET experiments were performed in Tris-polymix buffer [50 mM Tris-OAc (pH 25°C = 7.0), 100 mM KCl, 5 mM NH 4 OAc, 0.5 mM Ca(OAc) 2 , 0.1 mM EDTA, 10 mM β-mercaptoethanol, 5 mM putrescine-HCl, and 1 mM spermidine, free base] containing 15 mM Mg(OAc) 2 and supplemented with an oxygen-scavenging system (protocatechuic acid/protocatechuate-3,4-dioxygenase) (28) and a triplet-state quencher mixture [1 mM 1,3,5,7-cyclooctatetraene (Sigma-Aldrich) and 1 mM 3-nitrobenzyl alcohol (Fluka)] (29). Further details regarding data acquisition, processing, and analysis are in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

Ning

Fei

Gonzalez

2014

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

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One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines. During the catalytic cycle of many enzymes, multiple, spatially distant enzyme structural elements must often undergo functionally important conformational changes (1). Consistent with the view that such structural rearrangements must be rapidly organized and executed to maintain catalytic efficiency, many recent studies strongly suggest that small, monomeric protein enzymes have evolved complex networks of cooperative conformational changes that coordinate the inherently stochastic conformational fluctuations of multiple structural elements in a manner that is optimal for catalysis (2). Within the context of energy landscape theory (3), such enzymes can be thought of as having evolved dynamic energy landscapes that bias enzyme conformational sampling in such a manner to maximize the efficiency of catalysis (2). Related proposals suggest that binding of allosteric effectors to enzymes remodels such energy landscapes, altering enzyme conformational sampling as part of the mechanisms through which these effectors regulate enzymatic activity (4).Unfortunately, the conformational dynamics of large, macromolecular complexes remain much more challenging to characterize than those of small, monomeric proteins (5), making it very difficult to elucidate the role that cooperative conformational changes play in the function and regulation of biomolecular machines such as the ...

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“…Thiostreptonclass peptide antibiotics bind to the large ribosomal subunit GTPase-activating center to disrupt ternary complex binding to the A site (38). GE2270A class thiazolyl peptide antibiotics bind directly to EF-Tu at the domain 1/2 interface to prevent its interaction with aa-tRNA (36).…”

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confidence: 99%

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

Burnett

Altman

Ferrao

et al. 2013

Journal of Biological Chemistry

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Background: Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. Results: EF-Tu⅐GTP⅐aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). Conclusion: EF-Ts directly facilitates the formation and disassociation of ternary complex. Significance: This system demonstrates a novel function of EF-Ts.

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Thiostrepton inhibition of tRNA delivery to the ribosome

Cited by 48 publications

References 28 publications

The role of fluctuations in tRNA selection by the ribosome

The role of fluctuations in tRNA selection by the ribosome

The ribosome uses cooperative conformational changes to maximize and regulate the efficiency of translation

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

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