THE EFFECTS OF MULTI-SITE PHOSPHORYLATION ON THE STRUCTURE AND FUNCTION OF THE CARBOXYL-TERMINAL DOMAIN OF THE RNA POLYMERASE II LARGE SUBUNIT
Open Access
Author:
Gibbs, Eric Bryant
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
February 10, 2017
Committee Members:
Scott A. Showalter, Dissertation Advisor/Co-Advisor Scott A. Showalter, Committee Chair/Co-Chair Philip C. Bevilacqua, Committee Member William G. Noid, Committee Member David Gilmour, Outside Member
Keywords:
IDP RNAPII CTD Intrinsically Disordered Protein NMR SAXS P-TEFb Ssu72
Abstract:
Intrinsically disordered proteins (IDPs) are broadly defined as protein regions that do not cooperatively fold into spatially or temporally stable structures. A growing body of research supports the hypothesis that structural disorder renders IDPs uniquely capable of regulating key biological processes such as cellular signaling and transcription. Yet, this conformational plasticity often precludes the characterization of IDPs by traditional structural biology techniques. Advances in NMR spectroscopy, mass spectrometry, and small angle X-ray scattering presented here have enabled rigorous mechanistic studies of disordered proteins and regions, as exemplified by our investigation into the effects of multi-site phosphorylation on the structure and function of the carboxyl-terminal domain of the RNA polymerase II large subunit (CTD). We identify phosphorylation sites in the Drosophila melanogaster CTD that are targeted by the Positive Transcription Elongation Factor b (DmP-TEFb). We show that phosphorylation occurs primarily at Ser5 residues and that Tyr1 is necessary this specificity. Importantly, we demonstrate that Ser5 phosphorylation induces highly sequence-specific conformational switches in the CTD, which tune the apparent activity of CTD-interacting factors, using the CTD phosphatase Ssu72-Symplekin as an example. These studies highlight how regulation of IDPs can be mediated through cryptic sequence features and establish a foundation for elucidating the molecular basis of CTD regulation.
