Letter | Published:

Structure of RNA polymerase I transcribing ribosomal DNA genes

Nature volume 540, pages 607610 (22 December 2016) | Download Citation

Abstract

RNA polymerase I (Pol I) is a highly processive enzyme that transcribes ribosomal DNA (rDNA) and regulates growth of eukaryotic cells1,2,3,4. Crystal structures of free Pol I from the yeast Saccharomyces cerevisiae have revealed dimers of the enzyme stabilized by a ‘connector’ element and an expanded cleft containing the active centre in an inactive conformation5,6,7. The central bridge helix was unfolded and a Pol-I-specific ‘expander’ element occupied the DNA-template-binding site. The structure of Pol I in its active transcribing conformation has yet to be determined, whereas structures of Pol II and Pol III have been solved with bound DNA template and RNA transcript8,9,10. Here we report structures of active transcribing Pol I from yeast solved by two different cryo-electron microscopy approaches. A single-particle structure at 3.8 Å resolution reveals a contracted active centre cleft with bound DNA and RNA, and a narrowed pore beneath the active site that no longer holds the RNA-cleavage-stimulating domain of subunit A12.2. A structure at 29 Å resolution that was determined from cryo-electron tomograms of Pol I enzymes transcribing cellular rDNA confirms contraction of the cleft and reveals that incoming and exiting rDNA enclose an angle of around 150°. The structures suggest a model for the regulation of transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively.

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Acknowledgements

We thank O. Gadal and I. Lèger-Silvestre for technical assistance with the yeast Miller tree spreading technique, and H. Schwalbe for initial discussions. We thank T. Gubbey for initial experiments on the Pol I elongation complex and C. Bernecky, C. Plaschka and D. Tegunov for support with the single-particle data analysis. We thank T. Schulz for yeast fermentation. S.N. was supported by a PhD student fellowship from the Boehringer Ingelheim Fonds. P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB860, SPP1935), the Advanced Grant ‘TRANSREGULON’ from the European Research Council (grant agreement No 693023), and the Volkswagen Foundation. A.S.F. was supported by the Deutsche Forschungsgemeinschaft (SFB 902) and the Starting Grant ‘JTOMO’ from the European Research Council.

Author information

Author notes

    • Simon Neyer
    •  & Michael Kunz

    These authors contributed equally to this work.

    • Patrick Cramer
    •  & Achilleas S. Frangakis

    These authors jointly supervised this work.

Affiliations

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

    • Simon Neyer
    • , Merle Hantsche
    • , Christoph Engel
    •  & Patrick Cramer
  2. Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Goethe University Frankfurt, Max-von-Laue Str. 15, Frankfurt 60438, Germany

    • Michael Kunz
    • , Christian Geiss
    • , Victor-Valentin Hodirnau
    • , Anja Seybert
    • , Margot P. Scheffer
    •  & Achilleas S. Frangakis

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Contributions

S.N. planned and carried out the single particle sample preparation, data collection and data analysis. M.K. planned and carried out the tomographic data analysis. C.G. carried out the sample preparation for tomography. M.H. advised on structure determination procedures. V.V.H. advised on and carried out sample preparation for tomography. A.S. advised on sample preparation for tomography. C.E. advised on biochemical procedures. M.P.S. advised on tomographic data analysis. P.C. designed and supervised research, and supervised single particle structure determination. A.S.F. designed and supervised research, supervised single particle data collection and performed tomographic data collection and analysis. S.N., P.C. and A.S.F. prepared the manuscript, with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Patrick Cramer or Achilleas S. Frangakis.

Reviewer Information: Nature thanks R. Ebright, E. Nogales and E. Nudler for their contribution to the peer review of this work.

Extended data

Supplementary information

Videos

  1. 1.

    Conformational changes between free Pol I and Pol I EC

    The video illustrates the transition from free monomeric Pol I to elongating Pol I. The four major polymerase modules core, jaw-lobe, clamp and shelf are colored in grey, blue, yellow and pink, respectively. The movement of the rigid clamp-shelf module is highlighted together with the refolding of the bridge helix (green). The adjacent dimer of model of the crystal structure (PDB 4C2M) is shown briefly before the focus is set on the domain movements. Furthermore, the expander (light green), downstream DNA (blue), the DNA-RNA hybrid (blue-red) and the C-terminal domain of A12.2 are visualized.

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DOI

https://doi.org/10.1038/nature20561

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