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).

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 binding^{1, 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 bubble^{5, 6}. Notably, binding of dMyx stabilizes refolding of the '-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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