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Transcription inactivation through local refolding of the RNA polymerase structure

Nature volume 457, pages 332–335 (2009)Cite this article

Abstract

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become ‘frozen’ after inhibitor binding1,2. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism3. Here we report the structure of dMyx—a desmethyl derivative of myxopyronin B4—complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble5,6. Notably, binding of dMyx stabilizes refolding of the β′-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex—the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs.

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Figure 1: Structure of the RNAP–Myx complex.
Figure 2: Effect of RNAP mutations on dMyx activity.
Figure 3: A mechanism of the dMyx action.
Figure 4: Mutations in switch-2 affect the open complex formation.

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Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors have been in deposited in the PDB under accession number 3EQL.

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Acknowledgements

We thank T. Townes for critical reading of the manuscript and R. Saecker for many stimulating discussions. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Energy Research under contract No. W-31-109-Eng-38. This work was supported by National Institutes of Health grants to I.A. and D.G.V.

Author Contributions J.R.A., A.X.X., R.L. and S.E.W. synthesized the antibiotic. G.A.B. constructed, purified and analysed the properties of mutationally altered RNAPs. M.N.V. performed crystallization. M.N.V. and S.K. carried out data collection and processing. A.S. performed footprinting analysis. I.A. carried out vector construction, performed biochemical assays, and supervised functional analysis of the dMyx mechanism. E.N. contributed to data analysis. D.G.V. has determined, refined, analysed the structure and supervised the project. D.G.V. and I.A. jointly wrote the manuscript.

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Authors and Affiliations

  1. Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA,

    Georgiy A. Belogurov, Anastasiya Sevostyanova & Irina Artsimovitch

  2. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, 720 20th Street South, Birmingham, Alabama 35294, USA,

    Marina N. Vassylyeva, Sergiy Klyuyev & Dmitry G. Vassylyev

  3. Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, California 92121, USA ,

    James R. Appleman, Alan X. Xiang, Ricardo Lira & Stephen E. Webber

  4. Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, New York 10016, USA,

    Evgeny Nudler

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  1. Georgiy A. Belogurov
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Correspondence to Irina Artsimovitch or Dmitry G. Vassylyev.

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This file contains Supplementary Notes, Supplementary Table 1, Supplementary References and Supplementary Figures 1-12 with Legends (PDF 1879 kb)

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Belogurov, G., Vassylyeva, M., Sevostyanova, A. et al. Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457, 332–335 (2009). https://doi.org/10.1038/nature07510

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