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Nature 457, 332-335 (15 January 2009) | doi:10.1038/nature07510; Received 29 July 2008; Accepted 6 October 2008; Published online 22 October 2008

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Transcription inactivation through local refolding of the RNA polymerase structure

Georgiy A. Belogurov1, Marina N. Vassylyeva2, Anastasiya Sevostyanova1, James R. Appleman3, Alan X. Xiang3, Ricardo Lira3, Stephen E. Webber3, Sergiy Klyuyev2, Evgeny Nudler4, Irina Artsimovitch1 & Dmitry G. Vassylyev2

  1. Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA
  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
  3. Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, California 92121, USA
  4. Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, New York 10016, USA

Correspondence to: Irina Artsimovitch1Dmitry G. Vassylyev2 Correspondence and requests for materials should be addressed to I.A. (Email: artsimovitch.1@osu.edu) or D.G.V. (Email: dmitry@uab.edu).

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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 beta'-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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