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 visualization of key steps in human transcription initiation

Abstract

Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II (Pol II) at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. Here we have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed pre-initiation complex and the open-promoter complex, and to regulate start--initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, support a DNA translocation model of XPB and explain its essential role in promoter opening.

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: Stepwise assembly of the human PIC.
Figure 2: TFIIF engagement triggers a concerted conformational change in the PIC.
Figure 3: Stabilization of the PIC in the closed conformation by TFIIE.
Figure 4: Conformational rearrangements of the PIC upon promoter opening.
Figure 5: Positioning of TFIIH helicases and model of PIC assembly and promoter opening.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-2304 (TBP–TFIIA– TFIIB–DNA–Pol II), EMD-2305 (TBP–TFIIA–TFIIB–DNA–Pol II–TFIIF), EMD-2306 (TBP– TFIIA–TFIIB–DNA–Pol II–TFIIF–TFIIE) and EMD-2307 (TBP–TFIIA–TFIIB–DNA–Pol II– TFIIF–TFIIE in the OC mimic state). Negative stain EM density maps have been assigned accession numbers EMD-2308 (TBP–TFIIA–TFIIB–DNA–Pol II–TFIIF–TFIIE–TFIIH) and EMD-2309 (apo TFIIH).

References

  1. Matsui, T., Segall, J., Weil, P. A. & Roeder, R. G. Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J. Biol. Chem. 255, 11992–11996 (1980)

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Goodrich, J. A., Cutler, G. & Tjian, R. Contacts in context: promoter specificity and macromolecular interactions in transcription. Cell 84, 825–830 (1996)

    Article  CAS  PubMed  Google Scholar 

  4. Kornberg, R. D. The molecular basis of eukaryotic transcription. Proc. Natl Acad. Sci. USA 104, 12955–12961 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. 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  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Andel, F., III, Ladurner, A. G., Inouye, C., Tjian, R. & Nogales, E. Three-dimensional structure of the human TFIID-IIA-IIB complex. Science 286, 2153–2156 (1999)

    Article  CAS  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. Bernecky, C., Grob, P., Ebmeier, C. C., Nogales, E. & Taatjes, D. J. Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly. PLoS Biol. 9, e1000603 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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  ADS  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Bleichenbacher, M., Tan, S. & Richmond, T. J. Novel interactions between the components of human and yeast TFIIA/TBP/DNA complexes. J. Mol. Biol. 332, 783–793 (2003)

    Article  CAS  PubMed  Google Scholar 

  14. 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  PubMed  PubMed Central  Google Scholar 

  15. Sainsbury, S., Niesser, J. & Cramer, P. Structure and function of the initially transcribing RNA polymerase II–TFIIB complex. Nature 493, 437–440 (2013)

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. 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  PubMed  PubMed Central  Google Scholar 

  18. 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  PubMed  Google Scholar 

  19. Robert, F., Forget, D., Li, J., Greenblatt, J. & Coulombe, B. Localization of subunits of transcription factors IIE and IIF immediately upstream of the transcriptional initiation site of the adenovirus major late promoter. J. Biol. Chem. 271, 8517–8520 (1996)

    Article  CAS  PubMed  Google Scholar 

  20. Tyree, C. M. et al. Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II. Genes Dev. 7, 1254–1265 (1993)

    Article  CAS  PubMed  Google Scholar 

  21. Tan, S., Garrett, K. P., Conaway, R. C. & Conaway, J. W. Cryptic DNA-binding domain in the C terminus of RNA polymerase II general transcription factor RAP30. Proc. Natl Acad. Sci. USA 91, 9808–9812 (1994)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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  PubMed  PubMed Central  Google Scholar 

  23. Yan, Q., Moreland, R. J., Conaway, J. W. & Conaway, R. C. Dual roles for transcription factor IIF in promoter escape by RNA polymerase II. J. Biol. Chem. 274, 35668–35675 (1999)

    Article  CAS  PubMed  Google Scholar 

  24. Forget, D. et al. RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box. Proc. Natl Acad. Sci. USA 94, 7150–7155 (1997)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Orlicky, S. M., Tran, P. T., Sayre, M. H. & Edwards, A. M. Dissociable Rpb4-Rpb7 subassembly of rna polymerase II binds to single-strand nucleic acid and mediates a post-recruitment step in transcription initiation. J. Biol. Chem. 276, 10097–10102 (2001)

    Article  CAS  PubMed  Google Scholar 

  26. 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  PubMed  PubMed Central  Google Scholar 

  27. Buratowski, S., Sopta, M., Greenblatt, J. & Sharp, P. A. RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex. Proc. Natl Acad. Sci. USA 88, 7509–7513 (1991)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  29. 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  PubMed  Google Scholar 

  30. 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  PubMed  PubMed Central  Google Scholar 

  31. 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  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fernández-Tornero, C. et al. Conformational flexibility of RNA polymerase III during transcriptional elongation. EMBO J. 29, 3762–3772 (2010)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Cheung, A. C. & Cramer, P. Structural basis of RNA polymerase II backtracking, arrest and reactivation. Nature 471, 249–253 (2011)

    Article  ADS  CAS  PubMed  Google Scholar 

  34. Goodrich, J. A. & Tjian, R. Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. Cell 77, 145–156 (1994)

    Article  CAS  PubMed  Google Scholar 

  35. Conaway, R. C. & Conaway, J. W. General initiation factors for RNA polymerase II. Annu. Rev. Biochem. 62, 161–190 (1993)

    Article  CAS  PubMed  Google Scholar 

  36. 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. 37, 5803–5809 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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  PubMed  Google Scholar 

  38. Chakraborty, A. et al. Opening and closing of the bacterial RNA polymerase clamp. Science 337, 591–595 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gibbons, B. J. et al. Subunit architecture of general transcription factor TFIIH. Proc. Natl Acad. Sci. USA 109, 1949–1954 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Fan, L. et al. XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133, 789–800 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  42. Revyakin, A. et al. Transcription initiation by human RNA polymerase II visualized at single-molecule resolution. Genes Dev. 26, 1691–1702 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Juven-Gershon, T., Cheng, S. & Kadonaga, J. T. Rational design of a super core promoter that enhances gene expression. Nature Methods 3, 917–922 (2006)

    Article  CAS  PubMed  Google Scholar 

  44. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005)

    Article  CAS  PubMed  Google Scholar 

  45. Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    Article  CAS  PubMed  Google Scholar 

  47. Hohn, M. et al. SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 157, 47–55 (2007)

    Article  CAS  PubMed  Google Scholar 

  48. Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007)

    Article  CAS  PubMed  Google Scholar 

  49. Groft, C. M., Uljon, S. N., Wang, R. & Werner, M. H. Structural homology between the Rap30 DNA-binding domain and linker histone H5: implications for preinitiation complex assembly. Proc. Natl Acad. Sci. USA 95, 9117–9122 (1998)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  51. Knuesel, M. T., Meyer, K. D., Bernecky, C. & Taatjes, D. J. The human CDK8 subcomplex is a molecular switch that controls Mediator coactivator function. Genes Dev. 23, 439–451 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 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  PubMed  Google Scholar 

  53. Voss, N. R., Yoshioka, C. K., Radermacher, M., Potter, C. S. & Carragher, B. DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 166, 205–213 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Mallick, S. P., Carragher, B., Potter, C. S. & Kriegman, D. J. ACE: automated CTF estimation. Ultramicroscopy 104, 8–29 (2005)

    Article  CAS  PubMed  Google Scholar 

  55. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  PubMed  Google Scholar 

  56. Sorzano, C. O. et al. XMIPP: a new generation of an open-source image processing package for electron microscopy. J. Struct. Biol. 148, 194–204 (2004)

    Article  CAS  PubMed  Google Scholar 

  57. van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996)

    Article  CAS  PubMed  Google Scholar 

  58. Kostek, S. A. et al. Molecular architecture and conformational flexibility of human RNA polymerase II. Structure 14, 1691–1700 (2006)

    Article  CAS  PubMed  Google Scholar 

  59. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

    Article  CAS  PubMed  Google Scholar 

  60. Heymann, J. B. & Belnap, D. M. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007)

    Article  CAS  PubMed  Google Scholar 

  61. Lander, G. C. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  62. van Dijk, M. & Bonvin, A. M. 3D-DART: a DNA structure modelling server. Nucleic Acids Res. 37, W235–W239 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Lu, X. J. & Olson, W. K. 3DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structures. Nature Protocols 3, 1213–1227 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank C. Inouye for providing us with recombinant TFIIF and TFIIE; P. Grob and T. Houweling for electron microscopy and computer support, respectively; T. Goddard for help with Chimera; and members of the Nogales laboratory for technical advice on image processing. We are thankful to J. Kadonaga, J. Goodrich and M. Cianfrocco for their comments on the manuscript. We thank P. Cooper both for biochemical advice and for her comments on the manuscript. This work was funded by NIGMS (GM63072 to E.N.) and by NCI (CA127364 to D.J.T.). E.N. is a Howard Hughes Medical Institute Investigator.

Author information

Authors and Affiliations

Authors

Contributions

Y.H. designed and carried out the experiments; J.F. and D.J.T. provided essential reagents; Y.H. and E.N. analysed the data and wrote the paper.

Corresponding author

Correspondence to Eva Nogales.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-16 and Supplementary References. (PDF 2461 kb)

Step-wise assembly of the human transcription PIC as revealed by successive cryo-EM reconstructions

[1] TBP (red) - TFIIA (orange) - TFIIB (blue) - DNA (green, yellow and cyan) - Pol II (grey), [2] plus TFIIF (purple), [3] plus TFIIE (maroon). Docking of available atomic coordinates leads to pseudo-atomic models of these PIC subcomplexes (in the movie shown for the last reconstruction). The negative stain reconstruction of the PIC including TFIIH (light pink) allows for the localization of the TFIIH helicases XPD (green ribbon) and XPB (blue ribbon) relative to the rest of the complex. Comparison of the reconstructions for the closed pre-initiation complex and for the open promoter complex suggests that a DNA translocation activity of XPB plays an essential role in promoter opening. (MOV 32302 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

He, Y., Fang, J., Taatjes, D. et al. Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013). https://doi.org/10.1038/nature11991

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11991

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