A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription

Abstract

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

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Author details

  1. Matthew Allan Sdano

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6702-2755
  2. James M Fulcher

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9033-3623
  3. Sowmiya Palani

    Department of Radiation Oncology and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
  4. Mahesh B Chandrasekharan

    Department of Radiation Oncology and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
  5. Timothy J Parnell

    Department of Radiation Oncology and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
  6. Frank G Whitby

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
  7. Tim Formosa

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    For correspondence
    tim@biochem.utah.edu
    Competing interests
    Tim Formosa, Reviewing editor, eLife..
  8. Christopher P Hill

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    For correspondence
    chris@biochem.utah.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6796-7740

Funding

National Institutes of Health (R01GM116560)

  • Matthew Allan Sdano
  • Frank G Whitby
  • Tim Formosa
  • Christopher P Hill

National Institutes of Health (P50GM082545)

  • Matthew Allan Sdano
  • James M Fulcher
  • Frank G Whitby
  • Christopher P Hill

National Institutes of Health (P30CA042014)

  • Timothy J Parnell

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. John Kuriyan, University of California, Berkeley, United States

Publication history

  1. Received: May 17, 2017
  2. Accepted: August 11, 2017
  3. Accepted Manuscript published: August 16, 2017 (version 1)
  4. Version of Record published: September 14, 2017 (version 2)

Copyright

© 2017, Sdano et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Matthew Allan Sdano
  2. James M Fulcher
  3. Sowmiya Palani
  4. Mahesh B Chandrasekharan
  5. Timothy J Parnell
  6. Frank G Whitby
  7. Tim Formosa
  8. Christopher P Hill
(2017)
A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription
eLife 6:e28723.
https://doi.org/10.7554/eLife.28723
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