Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

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

et al. 2013

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107

The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-Å crystal structure of BlaS bound to a 70S·tRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3′ terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation.peptidyl transfer | ribosome crystal structure | termination inhibitor | translation inhibitor | translation termination T he growing problem of pathogen resistance to existing antibacterials prompts a search for alternative modes of inhibiting bacterial growth. The development of new drugs can be facilitated by understanding the mechanisms of action of known antibiotics (1). Because of its central role in cell metabolism, the ribosome is the target of numerous inhibitors that bind to various sites on the ribosome and interfere with different steps of protein synthesis. One of the predominant modes of action of ribosomal antibiotics is the inhibition of peptide bond formation. A majority of peptidyltransferase inhibitors, including the widely used antibacterials chloramphenicol, linezolid, and the lincosamides lincomycin and clindamycin, bind to the A site of the large ribosomal subunit at the peptidyl-transferase center of the ribosome (2-4). By contrast, blasticidin S (BlaS) binds to the P site of the large subunit (5) and inhibits the peptidyl-transferase reaction through a distinct mechanism, which is still poorly understood.BlaS, produced by some Streptomyces species, is a nucleoside analog consisting of a cytosine bonded to a pyranose ring and attached to an N-methyl-guanidine tail (Fig. 1A). BlaS has long been known to be a potent inhibitor of protein synthesis in bacteria and eukaryotes (6, 7). A crystal structure of the 50S subunit from Haloarcula marismortui bound to BlaS in the absence of tRNA revealed that BlaS occupies the P site of the large subunit (5). Two molecules of BlaS interact with the P loop and form base pairs with the universally conserved G2251 and G2252 of 23S ribosomal RNA (Escherichia coli 70S ribosome numbering is used throughout this article). These base pairing interactions closely mimic those of the two cytosine residues of the conserved CCA 3′ terminus of tRNA bound in the P site. Based on the finding that two molecules of BlaS mimic the cytosine residues of the...

Section: Resultssupporting

confidence: 63%

Blasticidin S inhibits translation by trapping deformed tRNA on the ribosome

Svidritskiy

Ling

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

et al. 2013

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107

“…In the absence of EF-G, tRNA fluctuations between classic and hybrid states on the 50S subunit, as well as intersubunit rotation, can occur spontaneously at rates comparable to rates of EF-G-catalyzed mRNA translocation (19,33,43,44). Therefore, it has been hypothesized that thermal energy is sufficient to drive tRNA and mRNA translocation upon spontaneous reverse rotation of the small subunit (15,32,(45)(46)(47). The comparison of pre-and posttranslocation states of EF-G-ribosome complex now reveals how spontaneous intersubunit rotation may be rectified into translocation by EF-G binding to the ribosome.…”

Section: Discussionmentioning

confidence: 99%

Structure of the ribosome with elongation factor G trapped in the pretranslocation state

Brilot

Коростелев

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

et al. 2013

Self Cite

135

219

Significance The ribosome decodes genetic information and synthesizes proteins in all living organisms. To translate the genetic information, the ribosome binds tRNA. During polypeptide chain elongation, the tRNA is moved together with the mRNA through the ribosome. This movement is called translocation and involves precisely coordinated steps that include binding of a protein called elongation factor G (EF-G). How exactly EF-G drives translocation is not fully understood. We show in this study a detailed three-dimensional molecular image of the ribosome bound to EF-G and two tRNAs, just before the tRNAs are translocated. The image provides mechanistic clues to how EF-G promotes tRNA translocation.

“…Spontaneous translocation is stimulated by a decrease in the concentration of magnesium ions, modification of ribosomal proteins with thiol‐specific reagents or removal of ribosomal proteins S12 and S13 . Furthermore, single‐round translocation can be induced by the peptidyl‐transferase inhibitors, sparsomycin, linkomycin, and chloramphenicol …”

Section: Fundamentals Of Ribosomal Translocationmentioning

confidence: 99%

“…The idea that EF‐G can promote translocation of tRNAs by biasing tRNA movements inside the ribosome is further supported by the phenomenon of antibiotic‐induced translocation. The antibiotics, sparsomycin, linkomycin and chloramphenicol, that bind to the A site of the large subunit were found to trigger mRNA and tRNA translocation on the small subunit . It has been hypothesized that these antibiotics induce translocation by sterically blocking the reverse, nonproductive movement of the peptidyl‐tRNA from the hybrid A/P to classical A/A state during spontaneous intersubunit rotation .…”

Section: The Molecular Mechanism Of Translocationmentioning

confidence: 99%

Structural insights into ribosome translocation

Ling

2016

WIREs RNA

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During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. Recent structural and single-molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the “head” domain of small ribosomal subunit undergoes forward- and back-swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF-G into the A site of the small ribosomal subunit that converts the thermally-driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF-G and tRNA during translocation is still not fully established and awaits further investigation.