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  • Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb ELIFE Mayfield, J. E., Irani, S., Escobar, E. E., Zhang, Z., Burkholder, N. T., Robinson, M. R., Mehaffey, M., Sipe, S. N., Yang, W., Prescott, N. A., Kathuria, K. R., Liu, Z., Brodbelt, J. S., Zhang, Y. 2019; 8

    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.

    View details for DOI 10.7554/eLife.48725

    View details for Web of Science ID 000483988000001

    View details for PubMedID 31385803

    View details for PubMedCentralID PMC6715403

  • Snapshots of C-S Cleavage in Egt2 Reveals Substrate Specificity and Reaction Mechanism CELL CHEMICAL BIOLOGY Irani, S., Naowarojna, N., Tang, Y., Kathuria, K. R., Wang, S., Dhembi, A., Lee, N., Yan, W., Lyu, H., Costello, C. E., Liu, P., Zhang, Y. 2018; 25 (5): 519-+

    Abstract

    Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.

    View details for DOI 10.1016/j.chembiol.2018.02.002

    View details for Web of Science ID 000432448700005

    View details for PubMedID 29503207

    View details for PubMedCentralID PMC5959753

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