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.

Structure and function of the initially transcribing RNA polymerase II–TFIIB complex

Abstract

The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II–TFIIB complex1,2 indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions3,4 enigmatic. Here we report crystal structures of the Pol II–TFIIB complex from the yeast Saccharomyces cerevisiae at 3.4 Å resolution and of an initially transcribing complex that additionally contains the DNA template and a 6-nucleotide RNA product. The structures reveal the entire B-reader and protein–nucleic acid interactions, and together with functional data lead to a more complete understanding of transcription initiation. TFIIB partially closes the polymerase cleft to position DNA and assist in its opening. The B-reader does not reach the active site but binds the DNA template strand upstream to assist in the recognition of the initiator sequence and in positioning the transcription start site. TFIIB rearranges active-site residues, induces binding of the catalytic metal ion B, and stimulates initial RNA synthesis allosterically. TFIIB then prevents the emerging DNA–RNA hybrid duplex from tilting, which would impair RNA synthesis. When the RNA grows beyond 6 nucleotides, it is separated from DNA and is directed to its exit tunnel by the B-reader loop. Once the RNA grows to 12–13 nucleotides, it clashes with TFIIB, triggering TFIIB displacement and elongation complex formation. Similar mechanisms may underlie all cellular transcription because all eukaryotic and archaeal RNA polymerases use TFIIB-like factors5, and the bacterial initiation factor sigma has TFIIB-like topology1,2 and contains the loop region 3.2 that resembles the B-reader loop in location, charge and function6,7,8. TFIIB and its counterparts may thus account for the two fundamental properties that distinguish RNA from DNA polymerases: primer-independent chain initiation and product separation from the template.

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

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 the Pol II–TFIIB complex at 3.4 Å resolution.
Figure 2: TFIIB changes Pol II domains and stimulates catalysis.
Figure 3: Structure of initially transcribing Pol II–TFIIB complex (ITC) elucidates RNA initiation and separation, and TFIIB release.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors of the Pol II–TFIIB complex and the ITC with 6-nucleotide RNA have been deposited in the Protein Data Bank under accession numbers 4BBR and 4BBS, respectively. Reprints and permissions information is available at www.nature.com/reprints.

References

  1. Kostrewa, D. et al. RNA polymerase II–TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009)

    CAS  PubMed  ADS  Article  Google Scholar 

  2. Liu, X., Bushnell, D. A., Wang, D., Calero, G. & Kornberg, R. D. Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism. Science 327, 206–209 (2010)

    CAS  PubMed  ADS  Article  Google Scholar 

  3. 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  PubMed  Article  Google Scholar 

  4. 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  PubMed  PubMed Central  Article  Google Scholar 

  5. Vannini, A. & Cramer, P. Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol. Cell 45, 439–446 (2012)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  ADS  Article  Google Scholar 

  7. 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  PubMed  ADS  Article  Google Scholar 

  8. Kulbachinskiy, A. & Mustaev, A. Region 3.2 of the sigma subunit contributes to the binding of the 3′-initiating nucleotide in the RNA polymerase active center and facilitates promoter clearance during initiation. J. Biol. Chem. 281, 18273–18276 (2006)

    CAS  PubMed  Article  Google Scholar 

  9. 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  PubMed  Article  Google Scholar 

  10. 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  PubMed  Article  Google Scholar 

  11. Grünberg, S., Warfield, L. & Hahn, S. Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nature Struct. Mol. Biol. 19, 788–796 (2012)

    Article  Google Scholar 

  12. Chen, Z. A. et al. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29, 717–726 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Eichner, J., Chen, H. T., Warfield, L. & Hahn, S. Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex. EMBO J. 29, 706–716 (2010)

    CAS  PubMed  Article  Google Scholar 

  14. Cabart, P., Ujvari, A., Pal, M. & Luse, D. S. Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes. Proc. Natl Acad. Sci. USA 108, 15786–15791 (2011)

    CAS  PubMed  ADS  Article  Google Scholar 

  15. Fishburn, J. & Hahn, S. Architecture of the yeast RNA polymerase II open complex and regulation of activity by TFIIF. Mol. Cell. Biol. 32, 12–25 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 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  PubMed  PubMed Central  Article  Google Scholar 

  17. Steitz, T. A., Smerdon, S. J., Jager, J. & Joyce, C. M. A unified polymerase mechanism for nonhomologous DNA and RNA polymerases. Science 266, 2022–2025 (1994)

    CAS  PubMed  ADS  Article  Google Scholar 

  18. Sosunov, V. et al. Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase. EMBO J. 22, 2234–2244 (2003)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 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  PubMed  ADS  Article  Google Scholar 

  20. 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)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  ADS  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Tran, K. & Gralla, J. D. Control of the timing of promoter escape and RNA catalysis by the transcription factor IIb fingertip. J. Biol. Chem. 283, 15665–15671 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 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  PubMed  Article  Google Scholar 

  25. Cheung, A. C., Sainsbury, S. & Cramer, P. Structural basis of initial RNA polymerase II transcription. EMBO J. 30, 4755–4763 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  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  PubMed  Article  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  PubMed  PubMed Central  Article  Google Scholar 

  29. Hawkes, N. A. & Roberts, S. G. The role of human TFIIB in transcription start site selection in vitro and in vivo. J. Biol. Chem. 274, 14337–14343 (1999)

    CAS  PubMed  Article  Google Scholar 

  30. Yang, C. & Ponticelli, A. S. Evidence that RNA polymerase II and not TFIIB is responsible for the difference in transcription initiation patterns between Saccharomyces cerevisiae and Schizosaccharomyces pombe. Nucleic Acids Res. 40, 6495–6507 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Sydow, J. F. et al. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol. Cell 34, 710–721 (2009)

    CAS  PubMed  Article  Google Scholar 

  32. Berrow, N. S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007)

    PubMed  PubMed Central  ADS  Article  Google Scholar 

  33. Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Seizl, M., Lariviere, L., Pfaffeneder, T., Wenzeck, L. & Cramer, P. Mediator head subcomplex Med11/22 contains a common helix bundle building block with a specific function in transcription initiation complex stabilization. Nucleic Acids Res. 39, 6291–6304 (2011)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank C. Bäjen, A. Cheung, S. Etzold, F. Hög, D. Kostrewa, N. Pirkl and other members of the Cramer laboratory. Part of this work was performed at the Swiss Light Source (SLS) at the Paul Scherrer Institut, Villigen, Switzerland. S.S. was supported by a postdoctoral fellowship from the Alexander-von-Humboldt Foundation. P.C. was supported by the Deutsche Forschungsgemeinschaft, SFB646, TR5, GraKo1721, SFB960, CIPSM, NIM, an Advanced Grant of the European Research Council, the LMUinnovativ project Bioimaging Network, the Jung-Stiftung, and the Vallee Foundation.

Author information

Authors and Affiliations

Authors

Contributions

S.S. carried out experiments except for functional analysis presented in Supplementary Fig. 5, which was carried out by J.N. P.C. initiated and supervised the project. S.S. and P.C. prepared the manuscript.

Corresponding author

Correspondence to Patrick Cramer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-5, Supplementary Table 1 and additional references. (PDF 5748 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sainsbury, S., Niesser, J. & Cramer, P. Structure and function of the initially transcribing RNA polymerase II–TFIIB complex. Nature 493, 437–440 (2013). https://doi.org/10.1038/nature11715

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11715

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

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