Letter | Published:

Transcription inactivation through local refolding of the RNA polymerase structure

Nature volume 457, pages 332335 (15 January 2009) | Download Citation


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Protein Data Bank

Data deposits

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


  1. 1.

    & Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation. Nature Struct. Mol. Biol. 15, 811–818 (2008)

  2. 2.

    et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007)

  3. 3.

    , , , & The myxopyronins, new inhibitors of bacterial RNA synthesis from Myxococcus fulvus (Myxobacterales). J. Antibiot. (Tokyo) 36, 1651–1658 (1983)

  4. 4.

    et al. Syntheses of novel myxopyronin B analogs as potential inhibitors of bacterial RNA polymerase. Bioorg. Med. Chem. Lett. 17, 6797–6800 (2007)

  5. 5.

    & The σ70 family of sigma factors. Genome Biol. 4, 203 (2003)

  6. 6.

    , & Distortion in the spacer region of Pm during activation of middle transcription of phage Mu. Proc. Natl Acad. Sci. USA 93, 9408–9413 (1996)

  7. 7.

    & DNA-melting at the Bacillus subtilis flagellin promoter nucleates near -10 and expands unidirectionally. J. Mol. Biol. 267, 47–59 (1997)

  8. 8.

    & Stimulation of open complex formation by nicks and apurinic sites suggests a role for nucleation of DNA melting in Escherichia coli promoter function. J. Biol. Chem. 273, 23558–23566 (1998)

  9. 9.

    , & Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004)

  10. 10.

    , , , & Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007)

  11. 11.

    , , , & Structural basis of transcription initiation: an RNA polymerase holoenzyme–DNA complex. Science 296, 1285–1290 (2002)

  12. 12.

    et al. DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA. J. Mol. Biol. 283, 741–756 (1998)

  13. 13.

    , , , & Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase. Proc. Natl Acad. Sci. USA 104, 7833–7838 (2007)

  14. 14.

    , & Two open complexes and a requirement for Mg2+ to open the λ PR transcription start site. Science 259, 358–361 (1993)

  15. 15.

    & A mutant RNA polymerase that forms unusual open promoter complexes. Proc. Natl Acad. Sci. USA 94, 13481–13486 (1997)

  16. 16.

    , & Structural basis of transcription: RNA polymerase II at 2.8 ångstrom resolution. Science 292, 1863–1876 (2001)

  17. 17.

    , , , & Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 Å resolution. Science 292, 1876–1882 (2001)

  18. 18.

    et al. Structural basis for converting a general transcription factor into an operon-specific virulence regulator. Mol. Cell 26, 117–129 (2007)

  19. 19.

    , , & Structure-based analysis of RNA polymerase function: the largest subunit’s rudder contributes critically to elongation complex stability and is not involved in the maintenance of RNA–DNA hybrid length. EMBO J. 21, 1369–1378 (2002)

  20. 20.

    et al. Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus. Acta Crystallogr. D 58, 1497–1500 (2002)

  21. 21.

    et al. Structural basis for transcription regulation by alarmone ppGpp. Cell 117, 299–310 (2004)

  22. 22.

    et al. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417, 712–719 (2002)

  23. 23.

    et al. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins. Cell 122, 351–363 (2005)

  24. 24.

    et al. Structural basis of transcription inhibition by antibiotic streptolydigin. Mol. Cell 19, 655–666 (2005)

  25. 25.

    et al. Structural basis for transcription inhibition by tagetitoxin. Nature Struct. Mol. Biol. 12, 1086–1093 (2005)

  26. 26.

    & Processing X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  27. 27.

    et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

  28. 28.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

  29. 29.

    MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)

  30. 30.

    Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta Crystallogr. D 55, 938–940 (1999)

  31. 31.

    & Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997)

Download references


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.

Author information


  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


  1. Search for Georgiy A. Belogurov in:

  2. Search for Marina N. Vassylyeva in:

  3. Search for Anastasiya Sevostyanova in:

  4. Search for James R. Appleman in:

  5. Search for Alan X. Xiang in:

  6. Search for Ricardo Lira in:

  7. Search for Stephen E. Webber in:

  8. Search for Sergiy Klyuyev in:

  9. Search for Evgeny Nudler in:

  10. Search for Irina Artsimovitch in:

  11. Search for Dmitry G. Vassylyev in:

Corresponding authors

Correspondence to Irina Artsimovitch or Dmitry G. Vassylyev.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Notes, Supplementary Table 1, Supplementary References and Supplementary Figures 1-12 with Legends

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.