Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis

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Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aatRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EFTu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.ribosome | error correction | fidelity | EF-Tu | ternary complex

Section: Why Did Mother Nature Evolve Two Proofreading Steps In Geneticmentioning

confidence: 98%

Two proofreading steps amplify the accuracy of genetic code translation

Ieong

Uzun

Selmer

et al. 2016

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“…Repeated in the elongation phase are (i) elongation factor thermo unstable (EF-Tu)-mediated accommodation of a codon-specified aminoacyl-tRNA to the A-site of the ribosome; (ii) transpeptidation from the P-site polypeptidyl n -tRNA ("n" specifies the number of amino acid residues) to the A-site aminoacyl-tRNA; and (iii) translocation of the polypeptidyl n+1 -tRNA back to the P-site facilitated by elongation factor EF-G. The elongation phase continues for the time window that is determined by the polypeptide size and the step time of ribosome progression, 40-70 ms in bacteria (1,2). However, nascent chain elongation does not necessarily proceed at a uniform speed through completion, and we still lack precise knowledge about the local elongation speed of individual proteins.…”

mentioning

confidence: 99%

Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

Chadani

Niwa

Chiba

et al. 2016

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.translation pausing | nascent polypeptides j Escherichia coli | peptidyl-tRNA

“…In this study we used rapid kinetics methods in a state-of-the-art in vitro translation system with in vivo-like rates (25,26) to characterize the mechanism of viomycin inhibition of the peptide elongation cycle. We constructed a quantitative model for viomycin action with precise estimates of the model parameters.…”

mentioning

confidence: 99%

Molecular mechanism of viomycin inhibition of peptide elongation in bacteria

Holm

Borg

Ehrenberg

et al. 2016

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Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steadystate kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.protein synthesis | viomycin | ribosome | antibiotics | tuberculosis T uberculosis (TB) is a global threat to human health, and over 9 million people contracted the disease in 2013 (1). The emergence of strains of Mycobacterium tuberculosis, the causative agent of TB, resistant to several first-and second-line antitubercular drugs is a significant concern. The tuberactinomycin antibiotics are bacterial protein synthesis inhibitors, commonly used as second-line drugs against multidrug-resistant TB. Viomycin was the first drug of this class to be discovered (2, 3). It is a cyclic pentapeptide that contains several nonstandard amino acids ( Fig. 1A) and is produced by a nonribosomal peptidyl transferase (4).Viomycin affects bacterial protein synthesis by inhibiting mRNA translocation (5) and causing misreading of the genetic code (6). The present study focuses on viomycin inhibition of translocation. During translocation the mRNA moves through the ribosome by one base triplet (codon), the peptidyl transfer RNA (tRNA) moves from the ribosomal A to P site, and the deacylated tRNA moves from the P to E site. Translocation is catalyzed by elongation factor G (EF-G) in a GTP hydrolysis-dependent manner (7) and occurs via formation of tRNA hybrid states (8), relative rotation of the ribosomal subunits (9), and movement of the L1 stalk (10). Bulk FRET, chemical footprinting, and single-molecule FRET experiments have shown that viomycin stabilizes the ribosome in a pretranslocation state with tRNAs in hybrid A/P and P/E configurations, rotated ribosomal subunits, and the L1 stalk in a closed conformation interacting with the P-site tRNA (11-16); this structure has been visualized by cryo-EM (17). However, a crystal structure of the viomycin-bound ribosome (18) and one single-molecule FRET study (19) have suggested stabilization of the tRNAs in the classical state.Recent crystal (18,20) and cryo-EM (17) structures of the viomycin-bound riboso...