Article | Published:

Transcription initiation complex structures elucidate DNA opening

Nature volume 533, pages 353358 (19 May 2016) | Download Citation

Abstract

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Data deposits

Three-dimensional cryo-EM density maps of OC1, OC2, OC2-focused, OC3, OC3-focused, OC4, OC4-focused, OC5, and CC have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-3375, EMD-3376, EMD-3377, EMD-3378, EMD-3379, EMD-3380, EMD-3381, EMD-3382, and EMD-3383, respectively. Coordinate files of the OC and CC have been deposited in the Protein Data Bank under accession numbers 5FYW and 5FZ5. Coordinates and structure factors of the Pol II-Tfg1 peptide and the Pol II-TFIIF crystals have been deposited at the Protein Data Bank under the accession numbers 5IP7 and 5IP9.

References

  1. 1.

    , , & Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549–561 (1989)

  2. 2.

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

  3. 3.

    & Structural insights into transcription initiation by RNA polymerase II. Trends Biochem. Sci. 38, 603–611 (2013)

  4. 4.

    , & Structural basis of transcription initiation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 129–143 (2015)

  5. 5.

    & DNA melting on yeast RNA polymerase II promoters. Science 261, 759–762 (1993)

  6. 6.

    & Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation. Nature 368, 160–163 (1994)

  7. 7.

    , & Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance. Genes Dev. 8, 515–524 (1994)

  8. 8.

    , & 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)

  9. 9.

    , & Promoter specificity of basal transcription factors. Cell 68, 1135–1144 (1992)

  10. 10.

    & 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)

  11. 11.

    , , & 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)

  12. 12.

    , , , & The requirement for the basal transcription factor IIE is determined by the helical stability of promoter DNA. EMBO J. 14, 810–819 (1995)

  13. 13.

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

  14. 14.

    , , , & Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism. Science 327, 206–209 (2010)

  15. 15.

    , & Structure and function of the initially transcribing RNA polymerase II–TFIIB complex. Nature 493, 437–440 (2013)

  16. 16.

    , & Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat. Struct. Mol. Biol. 19, 788–796 (2012)

  17. 17.

    et al. Architecture of an RNA polymerase II transcription pre-initiation complex. Science 342, 1238724 (2013)

  18. 18.

    et al. Conserved architecture of the core RNA polymerase II initiation complex. Nat. Commun. 5, 4310 (2014)

  19. 19.

    et al. Architecture of the RNA polymerase II-Mediator core initiation complex. Nature 518, 376–380 (2015)

  20. 20.

    , , & Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013)

  21. 21.

    et al. Structure of an RNA polymerase II preinitiation complex. Proc. Natl Acad. of Sci. USA 112, 13543–13548 (2015)

  22. 22.

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

  23. 23.

    , , & Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 272, 830–836 (1996)

  24. 24.

    , , & Crystal structure of a yeast TFIIA/TBP/DNA complex. Nature 381, 127–134 (1996)

  25. 25.

    , , & Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex. EMBO J. 29, 706–716 (2010)

  26. 26.

    & TFIIB and the regulation of transcription by RNA polymerase II. Chromosoma 116, 417–429 (2007)

  27. 27.

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

  28. 28.

    , , & 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)

  29. 29.

    , , , & 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)

  30. 30.

    , , , & Photo-cross-linking of a purified preinitiation complex reveals central roles for the RNA polymerase II mobile clamp and TFIIE in initiation mechanisms. Mol. Cell. Biol. 24, 1122–1131 (2004)

  31. 31.

    , & The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex. Nat. Struct. Mol. Biol. 14, 696–703 (2007)

  32. 32.

    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)

  33. 33.

    , & An extended winged helix domain in general transcription factor E/IIE alpha. J. Biol. Chem. 278, 48267–48274 (2003)

  34. 34.

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

  35. 35.

    et al. Structural insight into the TFIIE-TFIIH interaction: TFIIE and p53 share the binding region on TFIIH. EMBO J. 27, 1161–1171 (2008)

  36. 36.

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

  37. 37.

    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)

  38. 38.

    et al. Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39. eLife 4, e08378 (2015)

  39. 39.

    , , & Architecture of the RNA polymerase–Spt4/5 complex and basis of universal transcription processivity. EMBO J. 30, 1302–1310 (2011)

  40. 40.

    , & Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1421 (2000)

  41. 41.

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

  42. 42.

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

  43. 43.

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

  44. 44.

    & Structural basis for promoter-10 element recognition by the bacterial RNA polymerase σ subunit. Cell 147, 1257–1269 (2011)

  45. 45.

    et al. Structural basis of transcription initiation. Science 338, 1076–1080 (2012)

  46. 46.

    & Crystal structures of the E. coli transcription initiation complexes with a complete bubble. Mol. Cell 58, 534–540 (2015)

  47. 47.

    , , , & Structure of a bacterial RNA polymerase holoenzyme open promoter complex. eLife 4, (2015)

  48. 48.

    et al. Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies. Science 349, 882–885 (2015)

  49. 49.

    et al. Global regulation of promoter melting in naive lymphocytes. Cell 153, 988–999 (2013)

  50. 50.

    , & Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863–1876 (2001)

  51. 51.

    , & A conserved mediator hinge revealed in the structure of the MED7.MED21 (Med7.Srb7) heterodimer. J. Biol. Chem. 280, 18171–18178 (2005)

  52. 52.

    et al. A conserved GA element in TATA-less RNA polymerase II promoters. PLoS One 6, e27595 (2011)

  53. 53.

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

  54. 54.

    , , , & Computer controlled cryo-electron microscopy – TOM2 a software package for high-throughput applications. J. Struct. Biol. 175, 394–405 (2011)

  55. 55.

    , , , & Influence of electron dose rate on electron counting images recorded with the K2 camera. J. Struct. Biol. 184, 251–260 (2013)

  56. 56.

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

  57. 57.

    & CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

  58. 58.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  59. 59.

    et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

  60. 60.

    Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015)

  61. 61.

    et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  62. 62.

    et al. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47–52 (2015)

  63. 63.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004)

  64. 64.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010)

  65. 65.

    et al. Comparative protein structure modeling using Modeller. Current Protoc. Bioinformatics (2006)

  66. 66.

    , , & Crystal structure of a yeast TBP/TATA-box complex. Nature 365, 512–520 (1993)

  67. 67.

    et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12, 7–8 (2015)

  68. 68.

    , , & Structural and binding studies of the C-terminal domains of yeast TFIIF subunits Tfg1 and Tfg2. Proteins 80, 519–529 (2012)

  69. 69.

    et al. Consistent blind protein structure generation from NMR chemical shift data. Proc. Natl Acad. Sci. USA 105, 4685–4690 (2008)

  70. 70.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  71. 71.

    Using Situs for the integration of multi-resolution structures. Biophys. Rev. 2, 21–27 (2010)

  72. 72.

    et al. Structures of RNA polymerase II complexes with Bye1, a chromatin-binding PHF3/DIDO homologue. Proc. Natl Acad. Sci. USA 110, 15277–15282 (2013)

  73. 73.

    XDS. Acta Crystallogr. D 66, 125–132 (2010)

  74. 74.

    et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

  75. 75.

    , & Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution. J. Mol. Biol. 302, 1119–1127 (2000)

  76. 76.

    et al. Multiple functional domains of human transcription factor IIB: distinct interactions with two general transcription factors and RNA polymerase II. Genes Dev. 7, 1021–1032 (1993)

  77. 77.

    , & From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins. Trends Biochem. Sci. 38, 364–371 (2013)

  78. 78.

    et al. Mediator-dependent recruitment of TFIIH modules in preinitiation complex. Mol. Cell 31, 337–346 (2008)

  79. 79.

    & Inactivated RNA polymerase II open complexes can be reactivated with TFIIE. J. Biol. Chem. 287, 961–967 (2012)

Download references

Acknowledgements

We thank C. Bernecky, W. Mühlbacher, S. Neyer, S. Sainsbury, and D. Tegunov for help and discussions; L. Larivière and L. Wenzeck for cloning and initial purification of TFIIE; W. Mühlbacher for initial cloning of TFIIA; S. Bilakovic for the modified pET-DUET-1 vector; J. Mahamid for help with data collection for the CC; K. Maier for help with yeast growth assays; M. Raabe and H. Urlaub for protein identification; K. Kinkelin for initial Pol II–TFIIF co-crystallization; and S. Hahn for providing the TFA1 yeast strain and the shuffle plasmid pSH810. C.P. (SFB860), M.H. (GRK1721), and P.C. were supported by the Deutsche Forschungsgemeinschaft, the Advanced Grant TRANSIT of the European Research Council, and the Volkswagen Foundation.

Author information

Author notes

    • C. Plaschka
    •  & M. Hantsche

    These authors contributed equally to this work.

Affiliations

  1. Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany

    • C. Plaschka
    • , M. Hantsche
    • , C. Dienemann
    • , C. Burzinski
    •  & P. Cramer
  2. Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

    • J. Plitzko

Authors

  1. Search for C. Plaschka in:

  2. Search for M. Hantsche in:

  3. Search for C. Dienemann in:

  4. Search for C. Burzinski in:

  5. Search for J. Plitzko in:

  6. Search for P. Cramer in:

Contributions

C.P. designed and carried out high-resolution cryo-EM structure determinations of OC1–OC4. M.H. designed and carried out Pol II-TFIIF crystallographic analysis, and cryo-EM structure determinations of OC5 and CC. C.P. and M.H. designed and carried out functional assays. C.D. cloned and purified full-length TBP and TFIIA. C.D. and C.B. assisted with protein purification. J.P. supervised electron microscopy data collection. P.C. designed and supervised research. C.P., M.H., and P.C. prepared the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to P. Cramer.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature17990

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.