1. Biochemistry and Chemical Biology
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Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

  1. Joshua E Mayfield
  2. Seema Irani
  3. Edwin E Escobar
  4. Zhao Zhang
  5. Nathaniel T Burkholder
  6. Michelle R Robinson
  7. M Rachel Mehaffey
  8. Sarah N Sipe
  9. Wanjie Yang
  10. Nicholas A Prescott
  11. Karan Kathuria
  12. Zhijie Liu
  13. Jennifer Brodbelt
  14. Yan Zhang  Is a corresponding author
  1. University of Texas at Austin, United States
  2. University of Texas Health Science Center at San Antonio, United States
Research Article
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Cite this article as: eLife 2019;8:e48725 doi: 10.7554/eLife.48725

Abstract

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD’s coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

Article and author information

Author details

  1. Joshua E Mayfield

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Seema Irani

    Department of Chemical Engineering, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Edwin E Escobar

    Department of Chemistry, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0086-6264
  4. Zhao Zhang

    Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Nathaniel T Burkholder

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Michelle R Robinson

    Department of Chemistry, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. M Rachel Mehaffey

    Department of Chemistry, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Sarah N Sipe

    Department of Chemistry, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4554-7571
  9. Wanjie Yang

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Nicholas A Prescott

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0635-8906
  11. Karan Kathuria

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Zhijie Liu

    Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Diego, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Jennifer Brodbelt

    Department of Chemistry, University of Texas at Austin, Austin, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Yan Zhang

    Department of Molecular Biosciences, University of Texas at Austin, Austin, United States
    For correspondence
    jzhang@cm.utexas.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9360-5388

Funding

National Institute of General Medical Sciences (R01 GM104896)

  • Yan Zhang

National Institute of General Medical Sciences (R01 RM125882)

  • Jennifer Brodbelt

National Institute of Biomedical Imaging and Bioengineering (R21EB018391)

  • Jennifer Brodbelt

Welch Foundation (F-1778)

  • Yan Zhang

Welch Foundation (F-1155)

  • Jennifer Brodbelt

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

Reviewing Editor

  1. Jerry L Workman, Stowers Institute for Medical Research, United States

Publication history

  1. Received: May 23, 2019
  2. Accepted: August 5, 2019
  3. Accepted Manuscript published: August 6, 2019 (version 1)

Copyright

© 2019, Mayfield 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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