Article | Published:

Molecular structures of unbound and transcribing RNA polymerase III

Nature volume 528, pages 231236 (10 December 2015) | Download Citation

Abstract

Transcription of genes encoding small structured RNAs such as transfer RNAs, spliceosomal U6 small nuclear RNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. Here we present the cryo-electron microscopy structures of the Saccharomyces cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, which allow the building of a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82–C34–C31 heterotrimer in close proximity to the stalk. The C53–C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

The 3.9 Å cryo-EM map of elongating Pol III and the two maps of apo Pol III have been deposited in the Electron Microscopy Data Bank with accession codes EMD-3178 (elongating Pol III) and EMD-3179 (‘closed clamp’ apo Pol III) EMD-3180 (‘open clamp’ apo Pol III). The coordinates of the corresponding atomic models have been deposited in the Protein Data Bank under accession code 5fj8 (elongating Pol III) and 5fj9 (‘closed clamp’ apo Pol III), 5fja (‘open clamp’ apo Pol III).

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Acknowledgements

We thank Y. Sadian, H. Grötsch and R. Wetzel for help in Pol III purification. We are grateful to F. Thommen and M. Wahlers for set-up and maintenance of the high-performance computational environment of RELION. We acknowledge F. Schur, S. Fromm, C. Bertipaglia, M. Beck and G. Hofhaus for helpful advice regarding sample preparation and image processing. We also thank the ‘Fermentation et culture de microorganisms’ (IFR88, CNRS, Marseille). M.M.-M. and A.J.J. were supported by Marie-Sklodowska-Curie fellowships (FP7-PEOPLE-2011IEF301002, PIEF-GA-2012-331285). N.A.H. acknowledges support by the EMBL International PhD program, A.J.J. and J.K. by postdoctoral fellowships from the EMBL Interdisciplinary Postdoc Program (EIPOD) under Marie Curie COFUND actions (PCOFUND-GA-2008-229597) and C.W.M. by an ERC Advanced Grant (ERC-2013-AdG340964-POL1PIC).

Author information

Author notes

    • María Moreno-Morcillo
    •  & Sebastian Glatt

    Present addresses: Spanish National Cancer Research Centre (CNIO), Structural Biology and Biocomputing Programme, 28029 Madrid, Spain (M.M.-M.); Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7A str, 30-387 Krakow, Poland (S.G.).

Affiliations

  1. European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany

    • Niklas A. Hoffmann
    • , Arjen J. Jakobi
    • , María Moreno-Morcillo
    • , Sebastian Glatt
    • , Jan Kosinski
    • , Wim J. H. Hagen
    • , Carsten Sachse
    •  & Christoph W. Müller
  2. European Molecular Biology Laboratory (EMBL), Hamburg Unit, Notkestrasse 85, 22607 Hamburg, Germany

    • Arjen J. Jakobi

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Contributions

C.W.M. and C.S. initiated and supervised the project. N.A.H. established and performed Pol III experiments with input from C.W.M., S.G. and M.M.-M.; N.A.H., C.S. and W.J.H.H. established the grid freezing and acquisition parameters, N.A.H. and W.J.H.H. collected all cryo-EM data. N.A.H., M.M.-M. and A.J.J. built the initial model. N.A.H., A.J.J. and C.S. analysed cryo-EM data. J.K. performed homology modelling and alignments, A.J.J. established the refinement protocol. A.J.J. and N.A.H. optimized the refinement protocol and revised the models. N.A.H., A.J.J., S.G., M.M.-M., C.S. and C.W.M. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Carsten Sachse or Christoph W. Müller.

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https://doi.org/10.1038/nature16143

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