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Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening

Nature Structural & Molecular Biology volume 19, pages 788–796 (2012)Cite this article

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Abstract

Yeast RNA polymerase II (Pol II) general transcription factor TFIIE and the TFIIH subunit Ssl2 (yeast ortholog of mammalian XPB) function in the transition of the preinitiation complex (PIC) to the open complex. We show that the three TFIIE winged-helix (WH) domains form a heterodimer, with the Tfa1 (TFIIEα) WH binding the Pol II clamp and the Tfa2 (TFIIEβ) tandem WH domain encircling promoter DNA that becomes single-stranded in the open complex. Ssl2 lies adjacent to TFIIE, enclosing downstream promoter DNA. Unlike previous proposals, comparison of the PIC and open-complex models strongly suggests that Ssl2 promotes DNA opening by functioning as a double-stranded-DNA translocase, feeding 15 base pairs into the Pol II cleft. Right-handed threading of DNA through the Ssl2 binding groove, combined with the fixed position of upstream promoter DNA, leads to DNA unwinding and the open state.

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Figure 1: Functionally important regions in TFIIE.
Figure 2: Mapping of TFIIE-FeBABE cleavage in Pol II.
Figure 3: Cleavage of TFIIB by TFIIE-FeBABE in the PIC.
Figure 4: Position of TFIIE in the PIC.
Figure 5: Effect of mutations in the TFIIE dimerization, Pol II clamp and protrusion domains.
Figure 6: Orientation of the TFIIH subunit Ssl2 (XPB) in the PIC.
Figure 7: Protein cross-linking reveals Ssl2 interactions with TFIIE and TFIIB.
Figure 8: Comparison of the PIC and open-complex (OC) models suggests the role of Ssl2 in DNA strand opening.

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References

  1. Hahn, S. & Young, E.T. Transcriptional regulation in Saccharomyces cerevisiae: transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators. Genetics 189, 705–736 (2011).

    Article  CAS  Google Scholar 

  2. Thomas, M.C. & Chiang, C.M. The general transcription machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 41, 105–178 (2006).

    Article  CAS  Google Scholar 

  3. Wang, W., Carey, M. & Gralla, J.D. Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. Science 255, 450–453 (1992).

    Article  CAS  Google Scholar 

  4. Holstege, F.C.P., Fiedler, U. & Timmers, H.T.M. Three transitions in the RNA polymerase II transcription complex during initiation. EMBO J. 16, 7468–7480 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Pan, G. & Greenblatt, J. Initiation of transcription by RNA Polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site. J. Biol. Chem. 269, 30101–30104 (1994).

    CAS  PubMed  Google Scholar 

  12. Tantin, D. & Carey, M. A heteroduplex template circumvents the energetic requirement for ATP during activated transcription by RNA Pol II. J. Biol. Chem. 269, 17937–17400 (1994).

    Google Scholar 

  13. Holstege, F.C., van der Vliet, P.C. & Timmers, H.T. Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH. EMBO J. 15, 1666–1677 (1996).

    Article  CAS  Google Scholar 

  14. Ohkuma, Y. Multiple functions of general transcription factors TFIIE and TFIIH in transcription: possible points of regulation by trans-acting factors. J. Biochem. 122, 481–489 (1997).

    Article  CAS  Google Scholar 

  15. Feaver, W.J. et al. Yeast TFIIE. Cloning, expression, and homology to vertebrate proteins. J. Biol. Chem. 269, 27549–27553 (1994).

    CAS  PubMed  Google Scholar 

  16. Ohkuma, Y., Hashimoto, S., Wang, C.K., Horikoshi, M. & Roeder, R.G. Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-alpha. Mol. Cell Biol. 15, 4856–4866 (1995).

    Article  CAS  Google Scholar 

  17. Okuda, M. et al. A novel zinc finger structure in the large subunit of human general transcription factor TFIIE. J. Biol. Chem. 279, 51395–51403 (2004).

    Article  CAS  Google Scholar 

  18. Kuldell, N.H. & Buratowski, S. Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions. Mol. Cell Biol. 17, 5288–5298 (1997).

    Article  CAS  Google Scholar 

  19. Sakurai, H., Ohishi, T. & Fukasawa, T. Promoter structure–dependent functioning of the general transcription factor IIE in Saccharomyces cerevisiae. J. Biol. Chem. 272, 15936–15942 (1997).

    Article  CAS  Google Scholar 

  20. Aravind, L., Anantharaman, V., Balaji, S., Babu, M.M. & Iyer, L.M. The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol. Rev. 29, 231–262 (2005).

    Article  CAS  Google Scholar 

  21. Meinhart, A., Blobel, J. & Cramer, P. An extended winged helix domain in general transcription factor E/IIEα. J. Biol. Chem. 278, 48267–48274 (2003).

    Article  CAS  Google Scholar 

  22. Geiger, S.R. et al. RNA polymerase I contains a TFIIF-related DNA-binding subcomplex. Mol. Cell 39, 583–594 (2010).

    Article  CAS  Google Scholar 

  23. Okuda, M. et al. Structure of the central core domain of TFIIEβ with a novel double-stranded DNA-binding surface. EMBO J. 19, 1346–1356 (2000).

    Article  CAS  Google Scholar 

  24. Tanaka, A., Watanabe, T., Iida, Y., Hanaoka, F. & Ohkuma, Y. Central forkhead domain of human TFIIE beta plays a primary role in binding double-stranded DNA at transcription initiation. Genes Cells 14, 395–405 (2009).

    Article  CAS  Google Scholar 

  25. Shinkai, A. et al. The putative DNA-binding protein Sto12a from the thermoacidophilic archaeon Sulfolobus tokodaii contains intrachain and interchain disulfide bonds. J. Mol. Biol. 372, 1293–1304 (2007).

    Article  CAS  Google Scholar 

  26. Jawhari, A. et al. Structure and oligomeric state of human transcription factor TFIIE. EMBO Rep. 7, 500–505 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Luo, J., Fishburn, J., Hahn, S. & Ranish, J. An integrated chemical cross-linking and mass spectrometry approach to study protein complex architecture and function. Mol. Cell Proteomics 11, M111.008318 (2012).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Miller, G. & Hahn, S. A DNA-tethered cleavage probe reveals the path for promoter DNA in the yeast preinitiation complex. Nat. Struct. Mol. Biol. 13, 603–610 (2006).

    Article  CAS  Google Scholar 

  30. Grohmann, D. et al. The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation. Mol. Cell 43, 263–274 (2011).

    Article  CAS  Google Scholar 

  31. Grünberg, S., Bartlett, M.S., Naji, S. & Thomm, M. Transcription factor E is a part of transcription elongation complexes. J. Biol. Chem. 282, 35482–35490 (2007).

    Article  Google Scholar 

  32. Coin, F., Bergmann, E., Tremeau-Bravard, A. & Egly, J.M. Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH. EMBO J. 18, 1357–1366 (1999).

    Article  CAS  Google Scholar 

  33. Lin, Y.C., Choi, W.S. & Gralla, J.D. TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape. Nat. Struct. Mol. Biol. 12, 603–607 (2005).

    Article  CAS  Google Scholar 

  34. Datwyler, S.A. & Meares, C.F. Protein-protein interactions mapped by artificial proteases: where sigma factors bind to RNA polymerase. Trends Biochem. Sci. 25, 408–414 (2000).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. Liu, X., Bushnell, D.A., Silva, D.A., Huang, X. & Kornberg, R.D. Initiation complex structure and promoter proofreading. Science 333, 633–637 (2011).

    Article  CAS  Google Scholar 

  38. Douziech, M. et al. Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein-DNA photo-cross-linking. Mol. Cell Biol. 20, 8168–8177 (2000).

    Article  CAS  Google Scholar 

  39. Caruthers, J.M. & McKay, D.B. Helicase structure and mechanism. Curr. Opin. Struct. Biol. 12, 123–133 (2002).

    Article  CAS  Google Scholar 

  40. Goel, S., Krishnamurthy, S. & Hampsey, M. Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH. J. Biol. Chem. 287, 557–567 (2012).

    Article  CAS  Google Scholar 

  41. Chin, J.W. et al. An expanded eukaryotic genetic code. Science 301, 964–967 (2003).

    Article  CAS  Google Scholar 

  42. 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 

  43. 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).

    Article  CAS  Google Scholar 

  44. Werner, F. & Grohmann, D. Evolution of multisubunit RNA polymerases in the three domains of life. Nat. Rev. Microbiol. 9, 85–98 (2011).

    Article  CAS  Google Scholar 

  45. Vannini, A. & Cramer, P. Conservation between the RNA Polymerase I, II, and III Transcription Initiation Machineries. Mol. Cell 45, 439–446 (2012).

    Article  CAS  Google Scholar 

  46. Bischler, N. et al. Localization of the yeast RNA polymerase I–specific subunits. EMBO J. 21, 4136–4144 (2002).

    Article  CAS  Google Scholar 

  47. Wu, C.C., Lin, Y.C. & Chen, H.T. The TFIIF-like Rpc37/53 dimer lies at the center of a protein network to connect TFIIIC, Bdp1, and the RNA polymerase III active center. Mol. Cell Biol. 31, 2715–2728 (2011).

    Article  CAS  Google Scholar 

  48. Jennebach, S., Herzog, F., Aebersold, R. & Cramer, P. Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage. Nucleic Acids Res. published online, doi:10.1093/nar/gks220 (6 March 2012).

  49. Okamoto, T. et al. Analysis of the role of TFIIE in transcriptional regulation through structure-function studies of the TFIIEβ subunit. J. Biol. Chem. 273, 19866–19876 (1998).

    Article  CAS  Google Scholar 

  50. Brun, I., Sentenac, A. & Werner, M. Dual role of the C34 subunit of RNA polymerase III in transcription initiation. EMBO J. 16, 5730–5741 (1997).

    Article  CAS  Google Scholar 

  51. Vannini, A. et al. Molecular basis of RNA polymerase III transcription repression by Maf1. Cell 143, 59–70 (2010).

    Article  CAS  Google Scholar 

  52. Coin, F. et al. Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH. Nat. Genet. 20, 184–188 (1998).

    Article  CAS  Google Scholar 

  53. Lin, Y.C. & Gralla, J.D. Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE. Nucleic Acids Res. 33, 3072–3081 (2005).

    Article  CAS  Google Scholar 

  54. Treutlein, B. et al. Dynamic architecture of a minimal RNA polymerase II open promoter complex. Mol. Cell 46, 136–146 (2012).

    Article  CAS  Google Scholar 

  55. Söding, J., Biegert, A. & Lupas, A.N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005).

    Article  Google Scholar 

  56. Eswar, N., Eramian, D., Webb, B., Shen, M.Y. & Sali, A. Protein structure modeling with MODELLER. Methods Mol. Biol. 426, 145–159 (2008).

    Article  CAS  Google Scholar 

  57. Colovos, C. & Yeates, T.O. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 2, 1511–1519 (1993).

    Article  CAS  Google Scholar 

  58. Lüthy, R., Bowie, J.U. & Eisenberg, D. Assessment of protein models with three-dimensional profiles. Nature 356, 83–85 (1992).

    Article  Google Scholar 

  59. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    Article  Google Scholar 

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Acknowledgements

We thank J. Luo and J. Ranish (Institute for Systems Biology, Seattle, Washington) for sharing their unpublished TFIIE–cross-linking data, J. Fishburn for advice and help with TFIIE purification and transcription assays, A. Vannini for coordinates of the Rpc34 WH model, G. Smith for discussions and B. Knutson, I. Kamenova and M. Bartlett for comments on the manuscript. This work was supported by grant RO1GM053451 to S.H. and Forschungsstipendium GR 3776/2-1 of the Deutsche Forschungsgemeinschaft to S.G.

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    Sebastian Grünberg, Linda Warfield & Steven Hahn

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S.G. designed and performed all biochemical experiments except for the Bpa cross-linking, which was designed and performed by L.W.; S.G. and S.H. designed and performed Tfa1 and Tfa2 genetic assays, performed structure modeling and prepared the manuscript.

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Correspondence to Steven Hahn.

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Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 3323 kb)

Supplementary Movie 1

Overall structure of the PIC and open complex models. (MOV 6594 kb)

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Grünberg, S., Warfield, L. & Hahn, S. Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat Struct Mol Biol 19, 788–796 (2012). https://doi.org/10.1038/nsmb.2334

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