TY - JOUR TI - Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb AU - Mayfield, Joshua E AU - Irani, Seema AU - Escobar, Edwin E AU - Zhang, Zhao AU - Burkholder, Nathaniel T AU - Robinson, Michelle R AU - Mehaffey, M Rachel AU - Sipe, Sarah N AU - Yang, Wanjie AU - Prescott, Nicholas A AU - Kathuria, Karan R AU - Liu, Zhijie AU - Brodbelt, Jennifer S AU - Zhang, Yan A2 - Workman, Jerry L A2 - Cole, Philip A A2 - Workman, Jerry L A2 - Young, Nicolas VL - 8 PY - 2019 DA - 2019/08/06 SP - e48725 C1 - eLife 2019;8:e48725 DO - 10.7554/eLife.48725 UR - https://doi.org/10.7554/eLife.48725 AB - 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. KW - post-translational modification KW - P-TEFb KW - transcription KW - promoter-proximal pausing KW - phosphorylation KW - ultraviolet photodissociation mass spectrometry JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -