Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb

Joshua E. Mayfield, Seema Irani, Edwin E. Escobar, Zhao Zhang, Nathaniel T. Burkholder, Michelle R. Robinson, M. Rachel Mehaffey, Sarah N. Sipe, Wanjie Yang, Nicholas A. Prescott, Karan R. Kathuria, Zhijie Liu, Jennifer S. Brodbelt, Yan Zhang

Research output: Contribution to journalArticle


The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the 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.

Original languageEnglish (US)
Publication statusPublished - Aug 6 2019



  • biochemistry
  • chemical biology
  • human
  • P-TEFb
  • phosphorylation
  • post-translational modification
  • promoter-proximal pausing
  • transcription
  • ultraviolet photodissociation mass spectrometry

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Mayfield, J. E., Irani, S., Escobar, E. E., Zhang, Z., Burkholder, N. T., Robinson, M. R., ... Zhang, Y. (2019). Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb. eLife, 8. https://doi.org/10.7554/eLife.48725