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

Zhang, Y.; Mayfield, J. E.; Irani, S.; Escobar, E. E.; Zhang, Z.; Burkholder, N. T.; Robinson, M. R.; Mehaffey, M. R.; Sipe, S. N.; Yang, W.; Prescott, N. A.; Kathuria, K.; Liu, Z.; Brodbelt, J.

doi:10.1101/652214

bioRxiv · 10.1101/652214

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

Zhang, Y.·Mayfield, J. E.·Irani, S.·Escobar, E. E.·Zhang, Z.·Burkholder, N. T.·Robinson, M. R.·Mehaffey, M. R.·Sipe, S. N.·Yang, W.·Prescott, N. A.·Kathuria, K.·Liu, Z.·Brodbelt, J.

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Abstract

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of RNA polymerase IIs C-terminal domain (CTD) 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 a reduction of phosphorylated Ser2 and 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 CTDs 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.

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Zhang, Y., Mayfield, J. E., Irani, S., Escobar, E. E., Zhang, Z., Burkholder, N. T., Robinson, M. R., Mehaffey, M. R., Sipe, S. N., Yang, W., Prescott, N. A., Kathuria, K., Liu, Z., Brodbelt, J.. 2019-05-28. Tyr1 phosphorylation promotes the phosphorylation of Ser2 on the C-terminal domain of RNA polymerase II by P-TEFb. https://doi.org/10.1101/652214

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