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.

Structural basis for transcription elongation by bacterial RNA polymerase

Nature volume 448, pages 157–162 (2007)Cite this article

Abstract

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-Å resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the β′-subunit ‘lid’ loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

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

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Overall structure of the ttEC.
Figure 2: Nucleic acid structure in the ttEC.
Figure 3: Schematic drawing of the protein/nucleic acid contacts.
Figure 4: Protein–nucleic acid interactions in the ttEC structure.
Figure 5: dwDNA and RNA/DNA hybrid strand separation.

References

  1. Archambault, J. & Friesen, J. D. Genetics of eukaryotic RNA polymerases I, II, and III. Microbiol. Rev. 57, 703–724 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Cramer, P. Multisubunit RNA polymerases. Curr. Opin. Struct. Biol. 12, 89–97 (2002)

    Article  CAS  Google Scholar 

  3. 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 

  4. Kettenberger, H., Armache, K. J. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004)

    Article  CAS  Google Scholar 

  5. Wang, D., Bushnell, D. A., Westover, K. D., Kaplan, C. D. & Kornberg, R. D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127, 941–954 (2006)

    Article  CAS  Google Scholar 

  6. Westover, K. D., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell 119, 481–489 (2004)

    Article  CAS  Google Scholar 

  7. Westover, K. D., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science 303, 1014–1016 (2004)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  9. Mooney, R. A., Darst, S. A. & Landick, R. Sigma and RNA polymerase: an on-again, off-again relationship? Mol. Cell 20, 335–345 (2005)

    Article  CAS  Google Scholar 

  10. Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006)

    Article  ADS  Google Scholar 

  11. Revyakin, A., Liu, C., Ebright, R. H. & Strick, T. R. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139–1143 (2006)

    Article  ADS  CAS  Google Scholar 

  12. Temiakov, D. et al. Structural basis for substrate selection by t7 RNA polymerase. Cell 116, 381–391 (2004)

    Article  CAS  Google Scholar 

  13. Yin, Y. W. & Steitz, T. A. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 116, 393–404 (2004)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  16. Tahirov, T. H. et al. Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution. Nature 420, 43–50 (2002)

    Article  ADS  CAS  Google Scholar 

  17. Yin, Y. W. & Steitz, T. A. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298, 1387–1395 (2002)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  19. Vassylyev, D. G. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature doi:10.1038/nature05931 (this issue).

  20. 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 

  21. Kuznedelov, K., Korzheva, N., Mustaev, A. & Severinov, K. 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)

    Article  CAS  Google Scholar 

  22. Cheetham, G. M. & Steitz, T. A. Structure of a transcribing T7 RNA polymerase initiation complex. Science 286, 2305–2309 (1999)

    Article  CAS  Google Scholar 

  23. Jiang, M., Ma, N., Vassylyev, D. G. & McAllister, W. T. RNA displacement and resolution of the transcription bubble during transcription by T7 RNA polymerase. Mol. Cell 15, 777–788 (2004)

    Article  CAS  Google Scholar 

  24. Foster, J. E., Holmes, S. F. & Erie, D. A. Allosteric binding of nucleoside triphosphates to RNA polymerase regulates transcription elongation. Cell 106, 243–252 (2001)

    Article  CAS  Google Scholar 

  25. Gong, X. Q., Zhang, C., Feig, M. & Burton, Z. F. Dynamic error correction and regulation of downstream bubble opening by human RNA polymerase II. Mol. Cell 18, 461–470 (2005)

    Article  CAS  Google Scholar 

  26. Landick, R. NTP-entry routes in multi-subunit RNA polymerases. Trends Biochem. Sci. 30, 651–654 (2005)

    Article  CAS  Google Scholar 

  27. Huang, J., Brieba, L. G. & Sousa, R. Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation. Biochemistry 39, 11571–11580 (2000)

    Article  CAS  Google Scholar 

  28. Naryshkina, T., Kuznedelov, K. & Severinov, K. The role of the largest RNA polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid. J. Mol. Biol. 361, 634–643 (2006)

    Article  CAS  Google Scholar 

  29. Toulokhonov, I. & Landick, R. The role of the lid element in transcription by E. coli RNA polymerase. J. Mol. Biol. 361, 644–658 (2006)

    Article  CAS  Google Scholar 

  30. Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R. & Block, S. M. Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465 (2005)

    Article  ADS  CAS  Google Scholar 

  31. Artsimovitch, I. & Landick, R. Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release. Genes Dev. 12, 3110–3122 (1998)

    Article  CAS  Google Scholar 

  32. Yarnell, W. S. & Roberts, J. W. Mechanism of intrinsic transcription termination and antitermination. Science 284, 611–615 (1999)

    Article  ADS  CAS  Google Scholar 

  33. Santangelo, T. J. & Roberts, J. W. Forward translocation is the natural pathway of RNA release at an intrinsic terminator. Mol. Cell 14, 117–126 (2004)

    Article  CAS  Google Scholar 

  34. Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999)

    Article  CAS  Google Scholar 

  35. Navaza, J. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D Biol. Crystallogr. 57, 1367–1372 (2001)

    Article  CAS  Google Scholar 

  36. Kashkina, E. et al. Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations. Nucleic Acids Res. 34, 4036–4045 (2006)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. 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)

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  Google Scholar 

  42. 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 

  43. Afonine, P. V., Grosse-Kunstleve, R. W. & Adams, P. D. A robust bulk-solvent correction and anisotropic scaling procedure. Acta Crystallogr. D Biol. Crystallogr. 61, 850–855 (2005)

    Article  Google Scholar 

  44. Baker, D., Bystroff, C., Fletterick, R. J. & Agard, D. A. PRISM: topologically constrained phased refinement for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 49, 429–439 (1993)

    Article  CAS  Google Scholar 

  45. Vassylyev, D. G. et al. Atomic model of a pyrimidine dimer excision repair enzyme complexed with a DNA substrate: structural basis for damaged DNA recognition. Cell 83, 773–782 (1995)

    Article  CAS  Google Scholar 

  46. Matthews, B. W. & Czerwinski, E. W. Local scaling: a method to reduce systematic errors in isomorphous replacement and anomalous scattering measurements. Acta Crystallogr. A 31, 480–497 (1975)

    Article  ADS  Google Scholar 

  47. Wang, J. H. et al. Structure of a functional fragment of VCAM-1 refined at 1.9 Å resolution. Acta Crystallogr. D Biol. Crystallogr. 52, 369–379 (1996)

    Article  CAS  Google Scholar 

  48. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002)

    Article  Google Scholar 

  49. Weeks, C. M. & Miller, R. Optimizing Shake-and-Bake for proteins. Acta Crystallogr. D Biol. Crystallogr. 55, 492–500 (1999)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  51. Chlenov, M. et al. Structure and function of lineage-specific sequence insertions in the bacterial RNA polymerase β′ subunit. J. Mol. Biol. 353, 138–154 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Temiakov and M. Anikin for assistance in crystallization at the initial stage of the project. We are grateful to R. Landick for helpful discussions and critical reading of the manuscript. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Energy Research. This work was supported by NIH grants to D.G.V. and I.A.

Author Contributions D.G.V. determined and analysed the structure and guided the project. A.P. purified RNAP. M.N.V. and A.P performed crystallization and data collection. T.H.T assisted with data collection and analysis. I.A. contributed scaffold design and analysis. D.G.V. and I.A. jointly wrote the manuscript.

The atomic coordinates are deposited in the Protein Data Bank under accession number 2O5I

Author information

Author notes

  1. Correspondence and requests for materials should be addressed to D.G.V. (e-mail: dmitry@uab.edu).

Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Schools of Medicine and Dentistry, 402B Kaul Genetics Building, 720 20th Street South, Birmingham, Alabama 35294, USA,

    Dmitry G. Vassylyev, Marina N. Vassylyeva & Anna Perederina

  2. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Lied Transplant Center, 10737A, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-7696, USA,

    Tahir H. Tahirov

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

    Irina Artsimovitch

Authors
  1. Dmitry G. Vassylyev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marina N. Vassylyeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anna Perederina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tahir H. Tahirov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Irina Artsimovitch
    View author publications

    You can also search for this author in PubMed Google Scholar

Ethics declarations

Competing interests

The atomic coordinates are deposited in the Protein Data Bank under accession number 2O5I. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3 and Supplementary Figures 1-9 with Legends. (PDF 1907 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Vassylyev, D., Vassylyeva, M., Perederina, A. et al. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007). https://doi.org/10.1038/nature05932

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05932

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