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

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

doi:10.7554/elife.48725

Abstract

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.

Highlights

The C-terminal domain of the RPB1 subunit of RNA polymerase II (CTD) is composed of a speciesspecific number of repeats of the consensus amino acid heptad YSPTSPS (Jeronimo et al, 2016)
Positive Transcription Elongation Factor b (P-TEFb) was initially identified as a CTD kinase that controls the elongation potential of RNA polymerase II, is required for the majority of RNA polymerase II transcription, and is specific for Ser2 in vivo (Chao and Price, 2001; Marshall et al, 1996; Ni et al, 2004)
Investigations on the effect of CTD phosphorylations on P-TEFb specificity have revealed that Ser7 (Czudnochowski et al, 2012) and Ser5 do not alter its preference for Ser5

Summary

Introduction

The C-terminal domain of the RPB1 subunit of RNA polymerase II (CTD) is composed of a speciesspecific number of repeats of the consensus amino acid heptad YSPTSPS (arbitrarily numbered as Tyr, Ser, Pro, Thr, Ser, Pro, and Ser7) (Jeronimo et al, 2016). This suggests that living cells needs this two-phosphate code system in order for RNA polymerase II to progress and make copies of specific genes These results are a step forward in understanding the complex control mechanisms cells use to make proteins from their DNA. Our results reconcile the discrepancy of P-TEFb kinase activity in vitro and in cells, showing that Tyr phosphorylation can prime P-TEFb and alter its specificity to Ser. Our results reconcile the discrepancy of P-TEFb kinase activity in vitro and in cells, showing that Tyr phosphorylation can prime P-TEFb and alter its specificity to Ser2 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, resulting in a varied PTM landscape along the CTD allowing for diversified co-transcriptional signaling

Results

Discussion

Materials and methods

Funding Funder