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.

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

Abstract

To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II–B complex at 4.3 Å resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the ‘B-core’ domain that binds the wall at the end of the cleft. DNA is then opened with the help of the ‘B-linker’ that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the ‘B-reader’ that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Structure of Pol II–B complex.
Figure 2: Models of closed and open complexes.
Figure 3: B-linker and DNA opening.
Figure 4: B-reader and DNA start site scanning.
Figure 5: Initiation–elongation transition and PIC architecture.
Figure 6: Eukaryotic and bacterial transcription initiation complexes.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors of the complete Pol II–B complex crystal structure have been deposited with the Protein Data Bank under accession number 3K1F. The closed and open complex models can be downloaded from http://www.lmb.uni-muenchen.de/cramer.

References

  1. Roeder, R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 (1996)

    CAS  Google Scholar 

  2. Cramer, P. et al. Structure of eukaryotic RNA polymerases. Annu. Rev. Biophys. 37, 337–352 (2008)

    CAS  Google Scholar 

  3. Hahn, S. Structure and mechanism of the RNA polymerase II transcription machinery. Nature Struct. Mol. Biol. 11, 394–403 (2004)

    CAS  Google Scholar 

  4. Armache, K.-J., Kettenberger, H. & Cramer, P. Architecture of the initiation-competent 12-subunit RNA polymerase II. Proc. Natl Acad. Sci. USA 100, 6964–6968 (2003)

    CAS  Google Scholar 

  5. Bushnell, D. A. & Kornberg, R. D. Complete RNA polymerase II at 4.1-Å resolution: implications for the initiation of transcription. Proc. Natl Acad. Sci. USA 100, 6969–6973 (2003)

    CAS  Google Scholar 

  6. 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)

    CAS  Google Scholar 

  7. Bushnell, D. A., Westover, K. D., Davis, R. E. & Kornberg, R. D. Structural basis of transcription: an RNA polymerase II–TFIIB cocrystal at 4.5 Angstroms. Science 303, 983–988 (2004)

    CAS  Google Scholar 

  8. Chen, H. T. & Hahn, S. Binding of TFIIB to RNA polymerase II: mapping the binding site for the TFIIB zinc ribbon domain within the preinitiation complex. Mol. Cell 12, 437–447 (2003)

    CAS  Google Scholar 

  9. Nikolov, D. B. et al. Crystal structure of a TFIIB–TBP–TATA-element ternary complex. Nature 377, 119–128 (1995)

    CAS  Google Scholar 

  10. Ranish, J. A., Yudkovsky, N. & Hahn, S. Intermediates in formation and activity of the RNA polymerase II preinitiation complex: holoenzyme recruitment and a postrecruitment role for the TATA box and TFIIB. Genes Dev. 13, 49–63 (1999)

    CAS  Google Scholar 

  11. Pardee, T. S., Bangur, C. S. & Ponticelli, A. S. The N-terminal region of yeast TFIIB contains two adjacent functional domains involved in stable RNA polymerase II binding and transcription start site selection. J. Biol. Chem. 273, 17859–17864 (1998)

    CAS  Google Scholar 

  12. Cho, E. J. & Buratowski, S. Evidence that transcription factor IIB is required for a post-assembly step in transcription initiation. J. Biol. Chem. 274, 25807–25813 (1999)

    CAS  Google Scholar 

  13. Chen, H. T. & Hahn, S. Mapping the location of TFIIB within the RNA polymerase II transcription preinitiation complex: a model for the structure of the PIC. Cell 119, 169–180 (2004)

    CAS  Google Scholar 

  14. Edwards, A. M., Kane, C. M., Young, R. A. & Kornberg, R. D. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro . J. Biol. Chem. 266, 71–75 (1991)

    CAS  Google Scholar 

  15. Armache, K.-J., Mitterweger, S., Meinhart, A. & Cramer, P. Structures of complete RNA polymerase II and its subcomplex Rpb4/7. J. Biol. Chem. 280, 7131–7134 (2005)

    CAS  Google Scholar 

  16. Zhu, W. et al. The N-terminal domain of TFIIB from Pyrococcus furiosus forms a zinc ribbon. Nature Struct. Biol. 3, 122–124 (1996)

    CAS  Google Scholar 

  17. Pinto, I., Wu, W.-H., Na, J. G. & Hampsey, M. Characterization of sua7 mutations defines a domain of TFIIB involved in transcripiton start site selection in yeast. J. Biol. Chem. 269, 30569–30573 (1994)

    CAS  Google Scholar 

  18. Naji, S., Bertero, M. G., Spitalny, P., Cramer, P. & Thomm, M. Structure-function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement. Nucleic Acids Res. 36, 676–687 (2008)

    CAS  Google Scholar 

  19. 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)

    CAS  Google Scholar 

  20. Giardina, C. & Lis, J. T. DNA melting on yeast RNA polymerase II promoters. Science 261, 759–762 (1993)

    CAS  Google Scholar 

  21. Renfrow, M. B. et al. Transcription factor B contacts promoter DNA near the transcription start site of the archaeal transcription initiation complex. J. Biol. Chem. 279, 2825–2831 (2004)

    CAS  Google Scholar 

  22. Bartlett, M. S., Thomm, M. & Geiduschek, E. P. Topography of the euryarchaeal transcription initiation complex. J. Biol. Chem. 279, 5894–5903 (2004)

    CAS  Google Scholar 

  23. Naji, S., Grunberg, S. & Thomm, M. The RPB7 orthologue E′ is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation. J. Biol. Chem. 282, 11047–11057 (2007)

    CAS  Google Scholar 

  24. Kusser, A. G. et al. Structure of an archaeal RNA polymerase. J. Mol. Biol. 376, 303–307 (2008)

    CAS  Google Scholar 

  25. Hirata, A., Klein, B. J. & Murakami, K. S. The X-ray crystal structure of RNA polymerase from Archaea. Nature 451, 851–854 (2008)

    CAS  Google Scholar 

  26. Kassavetis, G. A., Letts, G. A. & Geiduschek, E. P. The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening. EMBO J. 20, 2823–2834 (2001)

    CAS  Google Scholar 

  27. Kuehner, J. N. & Brow, D. A. Quantitative analysis of in vivo initiator selection by yeast RNA polymerase II supports a scanning model. J. Biol. Chem. 281, 14119–14128 (2006)

    CAS  Google Scholar 

  28. Bangur, C. S., Pardee, T. S. & Ponticelli, A. S. Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein–TFIIB–DNA complexes. Mol. Cell. Biol. 17, 6784–6793 (1997)

    CAS  Google Scholar 

  29. Faitar, S. L., Brodie, S. A. & Ponticelli, A. S. Promoter-specific shifts in transcription initiation conferred by yeast TFIIB mutations are determined by the sequence in the immediate vicinity of the start sites. Mol. Cell. Biol. 21, 4427–4440 (2001)

    CAS  Google Scholar 

  30. Zhang, D. Y., Carson, D. J. & Ma, J. The role of TFIIB–RNA polymerase II interaction in start site selection in yeast cells. Nucleic Acids Res. 30, 3078–3085 (2002)

    CAS  Google Scholar 

  31. Li, Y., Flanagan, P. M., Tschochner, H. & Kornberg, R. D. RNA polymerase II initiation factor interactions and transcription start site selection. Science 263, 805–807 (1994)

    CAS  Google Scholar 

  32. Zhang, Z. & Dietrich, F. S. Mapping of transcription start sites in Saccharomyces cerevisiae using 5′ SAGE. Nucleic Acids Res. 33, 2838–2851 (2005)

    CAS  Google Scholar 

  33. Chen, B. S. & Hampsey, M. Functional interaction between TFIIB and the Rpb2 subunit of RNA polymerase II: implications for the mechanism of transcription initiation. Mol. Cell. Biol. 24, 3983–3991 (2004)

    CAS  Google Scholar 

  34. Kwapisz, M. et al. Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways. EMBO J. 27, 2411–2421 (2008)

    CAS  Google Scholar 

  35. Thiebaut, M. et al. Futile cycle of transcription initiation and termination modulates the response to nucleotide shortage in S. cerevisiae . Mol. Cell 31, 671–682 (2008)

    CAS  Google Scholar 

  36. Kuehner, J. N. & Brow, D. A. Regulation of a eukaryotic gene by GTP-dependent start site selection and transcription attenuation. Mol. Cell 31, 201–211 (2008)

    CAS  Google Scholar 

  37. Werner, F. & Weinzierl, R. O. Direct modulation of RNA polymerase core functions by basal transcription factors. Mol. Cell. Biol. 25, 8344–8355 (2005)

    CAS  Google Scholar 

  38. Andrecka, J. et al. Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex. Nucleic Acids Res. 10.1093/nar/gkp601 (20 July 2009)

  39. Pal, M., Ponticelli, A. S. & Luse, D. S. The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. Mol. Cell 19, 101–110 (2005)

    CAS  Google Scholar 

  40. Kim, T. K., Ebright, R. H. & Reinberg, D. Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1421 (2000)

    CAS  Google Scholar 

  41. Chen, H.-T., Warfield, L. & Hahn, S. The positions of TFIIF and TFIIE in the RNA polymerase II transcription initiation complex. Nature Struct. Mol. Biol. 14, 696–703 (2007)

    CAS  Google Scholar 

  42. Cramer, P. Common structural features of nucleic acid polymerases. Bioessays 24, 724–729 (2002)

    CAS  Google Scholar 

  43. Murakami, K. S., Masuda, S. & Darst, S. A. Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 1280–1284 (2002)

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  45. Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O. & Darst, S. A. Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science 296, 1285–1290 (2002)

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  47. Geszvain, K., Gruber, T. M., Mooney, R. A., Gross, C. A. & Landick, R. A hydrophobic patch on the flap-tip helix of E. coli RNA polymerase mediates σ70 region 4 function. J. Mol. Biol. 343, 569–587 (2004)

    CAS  Google Scholar 

  48. Severinov, K. et al. The sigma subunit conserved region 3 is part of “5′-face” of active center of Escherichia coli RNA polymerase. J. Biol. Chem. 269, 20826–20828 (1994)

    CAS  Google Scholar 

  49. 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)

    CAS  Google Scholar 

  50. Young, B. A., Gruber, T. M. & Gross, C. A. Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting. Science 303, 1382–1384 (2004)

    CAS  Google Scholar 

Download references

Acknowledgements

P.C. was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich SFB646, the Transregio 5, the Forschergruppe ‘Regulation und Mechanismen der Ribosomen-Biogenese’, the Nanosystems Initiative Munich (NIM), the Ernst-Jung-Stiftung, and the Fonds der chemischen Industrie. M.S. was supported by the Boehringer-Ingelheim-Fonds and Elitenetzwerk Bayern. Part of this work was performed at the ESRF at Grenoble, France, and at the Swiss Light Source (SLS) at the Paul Scherrer Institut, Villigen, Switzerland. M.T. and M.E.Z. were supported by the DFG Forschergruppe ‘Regulation und Mechanismen der Ribosomenbiogenese’. We thank members of the Cramer laboratory, in particular E. Lehmann and K. Maier, and members of the Thomm laboratory, in particular P. Decartes, W. Forster and F. Hirchaud. We thank K. Römer and the Römer-Stiftung for support.

Author Contributions D.K. carried out structure determination and modelling. M.E.Z. carried out archaeal biochemical assays. K.-J.A. prepared and measured the crystals and did the initial data processing. M.E.Z. and M.S. carried out yeast analysis. K.L. provided technical assistance. M.T. supervised the archaeal biochemical work. P.C. designed and supervised the project and prepared the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Patrick Cramer.

Supplementary information

Supplementary Information

This file contains Supplementary Methods and Data, Supplementary Table 1, Supplementary Figures 1-6 with Legends and Supplementary References. (PDF 3709 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kostrewa, D., Zeller, M., Armache, KJ. et al. RNA polymerase II–TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009). https://doi.org/10.1038/nature08548

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08548

Further reading

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

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