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.

  • Article
  • Published:

The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex

Abstract

We incorporated the non-natural photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa) into the RNA polymerase II (Pol II) surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photo-cross-linking of preinitiation complexes (PICs) with these Pol II derivatives and hydroxyl-radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9, while TFIIE cross-links to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains alter both Pol II–TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9 and TFIIB. Together with previous biochemical and structural studies, these findings illuminate the structural organization of the PIC and the network of protein-protein interactions involved in transcription start site selection.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Bpa incorporation into TFIIB and photo-cross-linking.
Figure 2: Photo-cross-linking confirms binding between the TFIIB core domain and the Rpb2 wall domain within the PIC.
Figure 3: TFIIF binds the Rpb2 lobe and protrusion domains within the PIC.
Figure 4: TFIIE interacts with the Rpb1 clamp domain within the PIC.
Figure 5: Rpb2 lobe and protrusion domain mutations alter Pol II–IIF interaction and confer an upstream shift in the transcription start site.
Figure 6: Site-specific hydroxyl-radical protein cleavage reveals the binding site for the TFIIF dimerization domain within the PIC.
Figure 7: Model of PIC assembly.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  4. Sun, Z.W. & Hampsey, M. Identification of the gene (SSU71/TFG1) encoding the largest subunit of transcription factor TFIIF as a suppressor of a TFIIB mutation in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 92, 3127–3131 (1995).

    Article  CAS  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. Ghazy, M.A., Brodie, S.A., Ammerman, M.L., Ziegler, L.M. & Ponticelli, A.S. Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II. Mol. Cell. Biol. 24, 10975–10985 (2004).

    Article  CAS  Google Scholar 

  7. Freire-Picos, M.A., Krishnamurthy, S., Sun, Z.W. & Hampsey, M. Evidence that the Tfg1/Tfg2 dimer interface of TFIIF lies near the active center of the RNA polymerase II initiation complex. Nucleic Acids Res. 33, 5045–5052 (2005).

    Article  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).

    Article  CAS  Google Scholar 

  9. 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).

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Sun, Z.W., Tessmer, A. & Hampsey, M. Functional interaction between TFIIB and the Rpb9 (Ssu73) subunit of RNA polymerase II in Saccharomyces cerevisiae. Nucleic Acids Res. 24, 2560–2566 (1996).

    Article  CAS  Google Scholar 

  12. Ziegler, L.M., Khaperskyy, D.A., Ammerman, M.L. & Ponticelli, A.S. Yeast RNA polymerase II lacking the Rpb9 subunit is impaired for interaction with transcription factor IIF. J. Biol. Chem. 278, 48950–48956 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Dvir, A. Promoter escape by RNA polymerase II. Biochim. Biophys. Acta 1577, 208–223 (2002).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. Cramer, P. et al. Architecture of RNA polymerase II and implications for the transcription mechanism. Science 288, 640–649 (2000).

    Article  CAS  Google Scholar 

  17. Bushnell, D.A. & Kornberg, R.D. Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription. Proc. Natl. Acad. Sci. USA 100, 6969–6973 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. 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 

  20. Kim, T.K. et al. Trajectory of DNA in the RNA polymerase II transcription preinitiation complex. Proc. Natl. Acad. Sci. USA 94, 12268–12273 (1997).

    Article  CAS  Google Scholar 

  21. Robert, F. et al. Wrapping of promoter DNA around the RNA polymerase II initiation complex induced by TFIIF. Mol. Cell 2, 341–351 (1998).

    Article  CAS  Google Scholar 

  22. 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 

  23. Kim, T.K., Ebright, R.H. & Reinberg, D. Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1422 (2000).

    Article  CAS  Google Scholar 

  24. Chung, W.H. et al. RNA polymerase II/TFIIF structure and conserved organization of the initiation complex. Mol. Cell 12, 1003–1013 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Kauer, J.C., Erickson-Viitanen, S., Wolfe, H.R. Jr. & DeGrado, W.F. p-Benzoyl-L-phenylalanine, a new photoreactive amino acid. Photolabeling of calmodulin with a synthetic calmodulin-binding peptide. J. Biol. Chem. 261, 10695–10700 (1986).

    CAS  PubMed  Google Scholar 

  27. Giuliodori, S. et al. A composite upstream sequence motif potentiates tRNA gene transcription in yeast. J. Mol. Biol. 333, 1–20 (2003).

    Article  CAS  Google Scholar 

  28. Warfield, L., Ranish, J.A. & Hahn, S. Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA. Genes Dev. 18, 1022–1034 (2004).

    Article  CAS  Google Scholar 

  29. Fishburn, J., Mohibullah, N. & Hahn, S. Function of a eukaryotic transcription activator during the transcription cycle. Mol. Cell 18, 369–378 (2005).

    Article  CAS  Google Scholar 

  30. Reeves, W.M. & Hahn, S. Targets of the Gal4 transcription activator in functional transcription complexes. Mol. Cell. Biol. 25, 9092–9102 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Tsai, F.T. & Sigler, P.B. Structural basis of preinitiation complex assembly on human pol II promoters. EMBO J. 19, 25–36 (2000).

    Article  CAS  Google Scholar 

  33. Hekmatpanah, D.S. & Young, R.A. Mutations in a conserved region of RNA polymerase II influence the accuracy of mRNA start site selection. Mol. Cell. Biol. 11, 5781–5791 (1991).

    Article  CAS  Google Scholar 

  34. Gaiser, F., Tan, S. & Richmond, T.J. Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution. J. Mol. Biol. 302, 1119–1127 (2000).

    Article  CAS  Google Scholar 

  35. Geiger, J.H., Hahn, S., Lee, S. & Sigler, P.B. Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 272, 830–836 (1996).

    Article  CAS  Google Scholar 

  36. Chen, H.T., Legault, P., Glushka, J., Omichinski, J.G. & Scott, R.A. Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription. Protein Sci. 9, 1743–1752 (2000).

    Article  CAS  Google Scholar 

  37. Bushnell, D.A., Bamdad, C. & Kornberg, R.D. A minimal set of RNA polymerase II transcription protein interactions. J. Biol. Chem. 271, 20170–20174 (1996).

    Article  CAS  Google Scholar 

  38. Berroteran, R.W., Ware, D.E. & Hampsey, M. The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations. Mol. Cell. Biol. 14, 226–237 (1994).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  40. 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  CAS  Google Scholar 

  41. 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 

  42. Sambrook, J., Fritsch, E.F. & Maniatis, T. in Molecular Cloning: A Laboratory Manual 2nd edn. 15.74–15.79 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1989).

    Google Scholar 

  43. 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  CAS  Google Scholar 

  44. Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Miller, B. Moorefield, N. Mohibullah, T. Young and other members of the Hahn laboratory for their comments and suggestions throughout the course of this work; J. Eichner (Fred Hutchinson Cancer Research Center) for assistance with TFIIF purification; P. Schultz, J. Chin and A. Cropp (The Scripps Research Institute) for the non-natural tRNA/aminoacyl-tRNA synthetase plasmid and advice on use of non-natural amino acids; and B. Moorefield and N. Mohibullah for comments on the manuscript. This work was supported by grant 5R01GM053451 from the US National Institutes of Health to S.H.

Author information

Authors and Affiliations

Authors

Contributions

L.W. modified the non-natural amino acid incorporation system and performed the experiment in Figure 1. H.-T.C. performed and designed the remaining experiments. S.H. supervised the study. H.-T.C. and S.H. wrote the manuscript.

Corresponding author

Correspondence to Steven Hahn.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Methods (PDF 1814 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, HT., 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). https://doi.org/10.1038/nsmb1272

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1272

This article is cited by

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