Phosphatases and prolyl-isomerase in the regulation of the C-terminal domain of eukaryotic RNA polymerase II

In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1.