Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution

Nature volume 417, pages 712–719 (2002)Cite this article

Abstract

In bacteria, the binding of a single protein, the initiation factor σ, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 Å resolution. In the structure, two amino-terminal domains of the σ subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of σ is near the outlet of the RNA-exit channel, about 57 Å from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Holoenzyme crystal structure.
Figure 2: Comparison between the holoenzyme and T. aquaticus core structures.
Figure 3: Organization and structure of the σ.
Figure 4: Individual σ domains and their interactions with the core.
Figure 5: Catalytic centre.
Figure 6: Models of the holoenzyme–nucleic acid complexes.

References

  1. Sweetser, D., Nonet, M. & Young, R. A. Prokaryotic and eukaryotic RNA polymerase have homologous core subunits. Proc. Natl Acad. Sci. USA 84, 1192–1196 (1987)

    Article  ADS  CAS  Google Scholar 

  2. Ebright, R. H. RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J. Mol. Biol. 304, 687–698 (2000)

    Article  CAS  Google Scholar 

  3. Burgess, R. R., Travers, A. A., Dunn, J. J. & Bautz, E. K. F. Factors stimulating transcription by RNA polymerase. Nature 221, 43–44 (1969)

    Article  ADS  CAS  Google Scholar 

  4. Gross, C., Lonetto, M. & Losick, R. in Transcriptional Regulation (eds McKnight, S. R. & Yamamoto, K. R.) 129–176 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992)

    Google Scholar 

  5. Record, M. T. J., Reznikoff, W., Craig, M., McQuade, K. & Schlax, P. in Escherichia coli and Salmonella (ed. Neidhart, F. C.) 792–820 (ASM, Washington DC, 1996)

    Google Scholar 

  6. von Hippel, P. An integrated model of the transcription complex in elongation, termination, and editing. Science 281, 660–665 (1998)

    Article  CAS  Google Scholar 

  7. Bar-Nahum, G. & Nudler, E. Isolation and characterization of σ70-retaining transcription elongation complexes from Escherichia coli. Cell 106, 443–451 (2001)

    Article  CAS  Google Scholar 

  8. Mukhopadhyay, K. et al. Translocation of σ70 with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell 106, 453–463 (2001)

    Article  CAS  Google Scholar 

  9. Craig, M. L. 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)

    Article  CAS  Google Scholar 

  10. Gross, C. et al. The functional and regulatory roles of sigma factors in transcription. Cold Spring Harbor Symp. Quant. Biol. 63, 141–155 (1998)

    Article  CAS  Google Scholar 

  11. Ishihama, A. Functional modulation of Escherichia coli RNA polymerase. Annu. Rev. Microbiol. 54, 499–518 (2000)

    Article  CAS  Google Scholar 

  12. Lonetto, M., Gribskov, M. & Gross, C. The σ70 family: sequence conservation and evolutionary relationships. J. Bacteriol. 174, 3843–3849 (1992)

    Article  CAS  Google Scholar 

  13. Dombroski, A. J., Walter, W. A., Record, M. T., Siegele, D. A. & Gross, C. A. Polypeptides containing highly conserved region of transcription initiation factor Σ 70 exhibit specificity of binding to promoter DNA. Cell 70, 501–512 (1992)

    Article  CAS  Google Scholar 

  14. Barne, K. A., Bown, J. A., Busby, S. J. W. & Minchin, S. D. Region 2.5 of the Escherichia coli RNA polymerase σ70 subunit is responsible for the recognition of the ‘extended - 10’ motif at promoters. EMBO J. 16, 4034–4040 (1997)

    Article  CAS  Google Scholar 

  15. Fenton, M. S., Lee, S. J. & Gralla, J. D. Escherichia coli promoter opening and - 10 recognition: mutational analysis of σ70. EMBO J. 19, 1130–1137 (2000)

    Article  CAS  Google Scholar 

  16. Marr, M. T. & Roberts, J. W. Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide. Science 276, 1258–1260 (1997)

    Article  CAS  Google Scholar 

  17. Huang, X., Lopez de Saro, F. J. & Helmann, J. D. Sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with - 10 region single-stranded DNA. Nucleic Acids Res. 25, 2603–2609 (1997)

    Article  CAS  Google Scholar 

  18. Sharp, M. et al. The interface of σ with core RNA polymerase is extensive, conserved, and functionally specialized. Genes Dev. 13, 3015–3026 (1999)

    Article  CAS  Google Scholar 

  19. Lesley, S. A. & Burgess, R. R. Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase. Biochemistry 28, 7728–7734 (1989)

    Article  CAS  Google Scholar 

  20. Owens, J. T. et al. Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease. Proc. Natl Acad. Sci. USA 95, 6021–6026 (1998)

    Article  ADS  CAS  Google Scholar 

  21. Zhang, G. et al. A crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell 98, 811–824 (1999)

    Article  CAS  Google Scholar 

  22. Cramer, P., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: RNA polymerase II at 2.8 Å resolution. Science 292, 1863–1876 (2001)

    Article  ADS  CAS  Google Scholar 

  23. Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 Å resolution. Science 292, 1876–1882 (2001)

    Article  ADS  CAS  Google Scholar 

  24. Korzheva, N. et al. A structural model of transcription elongation. Science 289, 619–625 (2000)

    Article  ADS  CAS  Google Scholar 

  25. Naryshkin, N., Revyakin, A., Kim, Y., Mekler, V. & Ebright, R. H. Structural organization of the RNA polymerase–promoter open complex. Cell 101, 601–611 (2000)

    Article  CAS  Google Scholar 

  26. Coulombe, B. & Burton, Z. F. DNA bending and wrapping around RNA polymerase: a “revolutionary” model describing transcriptional mechanisms. Microbiol. Mol. Biol. Rev. 63, 457–478 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Dombroski, A. J., Walter, W. A. & Gross, C. A. Amino-terminal amino acids modulate σ-factor DNA-binding activity. Genes Dev. 7, 2446–2455 (1993)

    Article  CAS  Google Scholar 

  28. Siegele, D. A., Hu, J. C., Walter, W. A. & Gross, C. A. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J. Mol. Biol. 206, 591–603 (1989)

    Article  CAS  Google Scholar 

  29. Gardella, T., Moyle, H. & Susskind, M. M. A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity. J. Mol. Biol. 206, 579–590 (1989)

    Article  CAS  Google Scholar 

  30. Malhotra, A., Severinova, E. & Darst, S. A. Crystal structure of a σ70 subunit fragment from E. coli RNA polymerase. Cell 87, 127–136 (1996)

    Article  CAS  Google Scholar 

  31. Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993)

    Article  CAS  Google Scholar 

  32. Arthur, T. M., Anthony, L. C. & Burgess, R. R. Mutational analysis of β′260–309, a σ70 binding site located on Escherichia coli core RNA polymerase. J. Biol. Chem. 275, 23113–23119 (2000)

    Article  CAS  Google Scholar 

  33. Young, B. A. et al. A coiled-coil from the RNA polymerase β′ subunit allosterically induces selective nontemplate strand binding by σ70. Cell 105, 935–944 (2001)

    Article  CAS  Google Scholar 

  34. Dieci, G. et al. A universally conserved region of the largest subunit participates in the active site of RNA polymerase III. EMBO J. 14, 3766–3776 (1995)

    Article  CAS  Google Scholar 

  35. Brautigam, C. A. & Steitz, T. A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr. Opin. Struct. Biol. 1, 54–63 (1998)

    Article  Google Scholar 

  36. Bown, J. A. et al. Organization of open complexes at Escherichia coli promoters. J. Biol. Chem. 274, 2263–2270 (1999)

    Article  CAS  Google Scholar 

  37. Owens, J. T. et al. Mapping the promoter DNA sites proximal to conserved regions of σ70 in an Escherichia coli RNA polymerase–lacUV5 open promoter complex. Biochemistry 37, 7670–7675 (1998)

    Article  CAS  Google Scholar 

  38. Lim, H. M., Lee, H. J., Roy, S. & Adhya, S. A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding. Proc. Natl Acad. Sci. USA 98, 14849–14852 (2001)

    Article  ADS  CAS  Google Scholar 

  39. Daube, S. S. & von Hippel, P. H. Interactions of Escherichia coli σ70 within the transcription elongation complex. Proc. Natl Acad. Sci. USA 96, 8390–8395 (1999)

    Article  ADS  CAS  Google Scholar 

  40. Vassylyeva, M. N. et al. Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus. Acta. Crystallogr. (submitted)

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

    Article  CAS  Google Scholar 

  42. Yeates, T. D. Detecting and overcoming crystal twinning. Methods Enzymol. 276, 344–358 (1997)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  44. Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628 (1994)

    Article  ADS  CAS  Google Scholar 

  45. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Merrit, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997)

    Article  Google Scholar 

Download references

Acknowledgements

We thank M. Yamamoto for assistance during the data collection at the SPring-8 synchrotron beam line, BL45. We are grateful to T. Yeates for discussions and advice concerning the merohedral twinning problem. This work was supported in part by a grant from the National Institutes of Health to S.B. and by a grant from the Organized Research Combination System of Science and Technology Agency (Japan) to S.Y.

Author information

Authors and Affiliations

  1. Cellular Signaling Laboratory, RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo, 679-5148, Japan

    Dmitry G. Vassylyev, Shun-ichi Sekine & Shigeyuki Yokoyama

  2. Structurome Research Group, RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo, 679-5148, Japan

    Dmitry G. Vassylyev, Shun-ichi Sekine, Marina N. Vassylyeva & Shigeyuki Yokoyama

  3. Department of Microbiology, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, New York, 11203, USA

    Oleg Laptenko, Jookyung Lee & Sergei Borukhov

  4. RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, 230-0045, Yokohama, Japan

    Marina N. Vassylyeva & Shigeyuki Yokoyama

  5. Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Shigeyuki Yokoyama

Authors
  1. Dmitry G. Vassylyev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shun-ichi Sekine
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oleg Laptenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jookyung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marina N. Vassylyeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sergei Borukhov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shigeyuki Yokoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dmitry G. Vassylyev.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Vassylyev, D., Sekine, Si., Laptenko, O. et al. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417, 712–719 (2002). https://doi.org/10.1038/nature752

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature752

This article is cited by

Comments

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.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing