Article | Published:

Structural basis of RNA polymerase III transcription initiation

Nature volume 553, pages 301306 (18 January 2018) | Download Citation

Abstract

RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4–4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.

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Acknowledgements

We thank N. Cronin at the Institute of Cancer Research for help with yeast fermentation; C. Richardson for the computing infrastructure; C. Plaschka (MRC-LMB, Cambridge) for advice during data processing; and the staff at beamline M03 of the Diamond Light Source synchrotron (UK) for help with EM data collection (EM15629, EM16599 and EM166601). We acknowledge support and the use of resources of iNEXT, in particular C. Sachse and W. Hagen for EM data collection at EMBL Heidelberg (PID1956 and 2180). G.A.-P. is a recipient of a Marie Sklodowska-Curie Intra-European Fellowship (EU project 655238). E.M. is supported by Cancer Research UK (CR-UK C12209/A16749). A.V. is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) New Investigator Award (BB/K014390/1), a Cancer Research UK Programme Foundation (CR-UK C47547/A21536) and a Wellcome Trust Investigator Award (200818/Z/16/Z).

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Affiliations

  1. The Institute of Cancer Research, London SW7 3RP, UK

    • Guillermo Abascal-Palacios
    • , Ewan Phillip Ramsay
    • , Fabienne Beuron
    • , Edward Morris
    •  & Alessandro Vannini

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Contributions

G.A.-P. carried out yeast fermentation, Pol III purification, Pol III PIC reconstitution, EM specimen preparation, EM data collection and processing, model building and refinement. E.P.R. carried out EM data collection and processing of the Bdp1Δ(355–372) Pol III PIC and helped with EM data processing and analysis. F.B. carried out cryo-EM sample preparation, screening and sample collection. E.M. helped during initial EM characterization and data collection. A.V. designed and supervised research, analysed the structural data and prepared the manuscript with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alessandro Vannini.

Reviewer Information Nature thanks R. Maraia and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Life Sciences Reporting Summary

Videos

  1. 1.

    Conformational changes upon PIC and open complex formation

    Morphing between cPol3 and OC-PIC cryo-EM structures shows the structural rearrangements occurring during PIC formation, which result in the lock of the Pol III clamp and the stabilisation of the downstream edge of the DNA bubble. The morphing and the video were generated using Chimera55. OC-PIC core subunits are depicted as grey molecular surfaces. The C82/C34/C31 subcomplex, stalk, TFIIIB subunits and C160 clamp helices are shown as ribbon and coloured as in Figure 1b.

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https://doi.org/10.1038/nature25441

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