Article | Published:

Near-atomic resolution visualization of human transcription promoter opening

Nature volume 533, pages 359365 (19 May 2016) | Download Citation

Abstract

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA–RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB.

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Data deposits

Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-8132 (OC), EMD-8133 (ITC), EMD-8134 (ITC(−IIS)), EMD-8135 (CC core), EMD-8136 (OC core), EMD-8137 (ITC core), EMD-8138 (ITC(−IIS) core), EMD-8131 (TFIIH core). Model coordinates have been deposited in the Protein Data Bank (PDB) under accession numbers 5IY6 (CC), 5IY7 (OC), 5IY8 (ITC), 5IY9 (ITC(−IIS)), 5IYA (CC core), 5IYB (OC core), 5IYC (ITC core), 5IYD (ITC(−IIS) core), 5IVW (TFIIH core).

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Acknowledgements

We thank S. Zheng for providing XPB mAb, D. King for providing peptides, P. Grob and T. Houweling for electron microscopy and computer support, respectively; S. Scheres for advice on image processing, S. Kassube for providing TFIIS, and G. Cardone for advice concerning filtering according to local resolution. This work was funded by NIGMS (GM63072 to E.N. and GM110387 to I.I.) and the National Science Foundation (MCB-1149521 to I.I.). Computational resources were provided in part by XSEDE (CHE110042) and the National Energy Research for Scientific Computing Center (DE-AC02-05CH11231). E.N. and R.T are Howard Hughes Medical Institute Investigators.

Author information

Affiliations

  1. Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Yuan He
    •  & Eva Nogales
  2. Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA

    • Yuan He
  3. Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302, USA

    • Chunli Yan
    •  & Ivaylo Ivanov
  4. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

    • Jie Fang
    • , Robert Tjian
    •  & Eva Nogales
  5. Li Ka Shing Center for Biomedical and Health Sciences, University of California, Berkeley, California 94720, USA

    • Carla Inouye
    •  & Robert Tjian
  6. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

    • Robert Tjian
    •  & Eva Nogales

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Contributions

Y.H. designed and carried out the experiments; C.Y. and I.I. performed structural modelling; C.I., J.F. and Y.H. purified GTFs and Pol II; Y.H. and E.N. analysed the data and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Yuan He or Eva Nogales.

Extended data

Supplementary information

Videos

  1. 1.

    Cryo-EM reconstruction and MDFF model of core ITC

    Densities are shown as a semi-transparent surface following a similar colour scheme to that in the main section. A region corresponding to the double-psi beta-barrel domain composing the conserved core of Pol II within Rpb1 and Rpb2 is shown as an example of near-atomic resolution features (as in Fig. 2). Colour scheme for the promoter DNA is shown at the bottom.

  2. 2.

    Cryo-EM reconstruction and MDFF model of core ITC(-IIS)

    Densities are shown as a semi-transparent surface following a similar colour scheme to that in the main section. A region corresponding to the active site of Pol II is shown as an example of near-atomic resolution features (as in Fig. 2). Color scheme for the promoter DNA is shown at the bottom.

  3. 3.

    Transitions of nucleic acids through the transcription initiation process

    By aligning the three models of holo-PICs (CC, OC, and ITC) using the rigid part of Pol II, sequential states are morphed with a special focus on the nucleic acids regions.

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https://doi.org/10.1038/nature17970

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