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      NusG-Dependent RNA Polymerase Pausing and Tylosin-Dependent Ribosome Stalling Are Required for Tylosin Resistance by Inducing 23S rRNA Methylation in Bacillus subtilis

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          Antibiotic resistance is a growing health concern. Resistance mechanisms have evolved that provide bacteria with a growth advantage in their natural habitat such as the soil. We determined that B. subtilis, a Gram-positive soil organism, has a mechanism of resistance to tylosin, a macrolide antibiotic commonly used in the meat industry. Tylosin induces expression of yxjB, which encodes an enzyme that methylates 23S rRNA. YxjB-dependent methylation of 23S rRNA confers tylosin resistance. NusG-dependent RNA polymerase pausing and tylosin-dependent ribosome stalling induce yxjB expression, and hence tylosin resistance, by preventing transcription termination upstream of the yxjB coding sequence and by preventing repression of yxjB translation.


          Macrolide antibiotics bind to 23S rRNA within the peptide exit tunnel of the ribosome, causing the translating ribosome to stall when an appropriately positioned macrolide arrest motif is encountered in the nascent polypeptide. Tylosin is a macrolide antibiotic produced by Streptomyces fradiae. Resistance to tylosin in S. fradiae is conferred by methylation of 23S rRNA by TlrD and RlmA II. Here, we demonstrate that yxjB encodes RlmA II in Bacillus subtilis and that YxjB-specific methylation of 23S rRNA in the peptide exit tunnel confers tylosin resistance. Growth in the presence of subinhibitory concentrations of tylosin results in increased rRNA methylation and increased resistance. In the absence of tylosin, yxjB expression is repressed by transcription attenuation and translation attenuation mechanisms. Tylosin-dependent induction of yxjB expression relieves these two repression mechanisms. Induction requires tylosin-dependent ribosome stalling at an RYR arrest motif at the C terminus of a leader peptide encoded upstream of yxjB. Furthermore, NusG-dependent RNA polymerase pausing between the leader peptide and yxjB coding sequences is essential for tylosin-dependent induction. Pausing synchronizes the position of RNA polymerase with ribosome position such that the stalled ribosome prevents transcription termination and formation of an RNA structure that sequesters the yxjB ribosome binding site. On the basis of our results, we are renaming yxjB as tlrB.

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          Most cited references 29

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          Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria.

          Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in vivo activity of all such regulators in parallel and enables rapid screening for ribo-regulators that respond to metabolites of choice. Using this approach, we detected numerous antibiotic-responsive ribo-regulators that control antibiotic resistance genes in pathogens and in the human microbiome. Studying one such regulator in Listeria monocytogenes revealed an attenuation mechanism mediated by antibiotic-stalled ribosomes. Our results expose broad roles for conditional termination in regulating antibiotic resistance and provide a tool for discovering riboswitches and attenuators that respond to previously unknown ligands.
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            The regulatory roles and mechanism of transcriptional pausing.

            The multisubunit RNAPs (RNA polymerases) found in all cellular life forms are remarkably conserved in fundamental structure, in mechanism and in their susceptibility to sequence-dependent pausing during transcription of DNA in the absence of elongation regulators. Recent studies of both prokaryotic and eukaryotic transcription have yielded an increasing appreciation of the extent to which gene regulation is accomplished during the elongation phase of transcription. Transcriptional pausing is a fundamental enzymatic mechanism that underlies many of these regulatory schemes. In some cases, pausing functions by halting RNAP for times or at positions required for regulatory interactions. In other cases, pauses function by making RNAP susceptible to premature termination of transcription unless the enzyme is modified by elongation regulators that programme efficient gene expression. Pausing appears to occur by a two-tiered mechanism in which an initial rearrangement of the enzyme's active site interrupts active elongation and puts RNAP in an elemental pause state from which additional rearrangements or regulator interactions can create long-lived pauses. Recent findings from biochemical and single-molecule transcription experiments, coupled with the invaluable availability of RNAP crystal structures, have produced attractive hypotheses to explain the fundamental mechanism of pausing.
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              Geometric nomenclature and classification of RNA base pairs


                Author and article information

                Role: Editor
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                12 November 2019
                Nov-Dec 2019
                : 10
                : 6
                [a ]Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA
                [b ]Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA
                [c ]RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA
                University of Minnesota Medical School
                Author notes
                Address correspondence to Paul Babitzke, pxb28@ .

                Present address: Brandon L. Mouery, Curriculum in Genetics and Molecular Biology, the University of North Carolina, Chapel Hill, North Carolina, USA.

                H.Y. and A.V.Y. contributed equally to this article.

                Copyright © 2019 Yakhnin et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                supplementary-material: 7, Figures: 6, Tables: 2, Equations: 0, References: 33, Pages: 14, Words: 8697
                Funded by: NIH/NCI Intramural Research Program;
                Award Recipient :
                Funded by: HHS | National Institutes of Health (NIH),;
                Award ID: GM098399
                Award Recipient :
                Research Article
                Molecular Biology and Physiology
                Custom metadata
                November/December 2019


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