1. Structural Biology and Molecular Biophysics
  2. Chromosomes and Gene Expression

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

  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  Is a corresponding author
  8. Christopher P Hill  Is a corresponding author
  1. University of Utah School of Medicine, United States
https://doi.org/10.7554/eLife.28723
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Data availability

The following data sets were generated

Article and author information

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

Version 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.

Metrics

  • 2,071
    views
  • 403
    downloads
  • 61
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Share this article

https://doi.org/10.7554/eLife.28723

Categories and tags

Research organism

Further reading

    1. Structural Biology and Molecular Biophysics

    The mechanism of mammalian proton-coupled peptide transporters

    Simon M Lichtinger, Joanne L Parker ... Philip C Biggin
    Research Article

    Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

    1. Structural Biology and Molecular Biophysics

    Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation

    Hitendra Negi, Aravind Ravichandran ... Ranabir Das
    Research Article

    The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Special Issue: Allosteric regulation of kinase activity

    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.