Structural basis of RNA Polymerase II C-terminal domain kinase and phosphatase specificity and their impact on transcriptional regulation

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2020-01-30

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Burkholder, Nathaniel Tate

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Abstract

Transcription from a most basic perspective is the process of generating strands of RNA from DNA templates. However, in order to control when, where, and how much of specific RNAs are made, cells have evolved vast arrays of transcriptional regulatory mechanisms that allow for extensive differentiation and formation of complex traits. One of the unique and most important mechanisms of transcriptional regulation in eukaryotic cells is the reversible phosphorylation of the RNA polymerase II C-terminal domain (RNAPII CTD). The CTD contains heptad repeats composed of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 and all of the non-proline sites are phosphorylated in cells. The human CTD contains 52 repeats where the first 26 proximal heptads are mostly consensus sequence whereas the last 26 distal heptads contain several variations primarily at the Ser7 position. In Chapter 2, I describe how these variations and their modifications alter the phosphorylation of Tyr1 sites by using a combination of biochemical assays and mass spectrometry. Data presented in this chapter reveal how a conserved positively charged pocket in tyrosine kinases likely mediates the interaction residues in the Ser7 position and can potentially affect in vivo Tyr1 phospho-patterning. Futhermore, in Chapter 3 I describe the methodology behind synthesis and testing of cis/trans-locked Ser-Pro CTD peptides for understanding the role of prolyl isomerization on CTD regulation. We used these tools to determine the specificity of several CTD phosphatases, which revealed how the Ser5 phosphatase SSU72 structurally prefers the cis- over the trans-configuration of the phosphorylated Ser5-Pro6 motif.

Among the phosphatases discovered to dephosphorylate the CTD, the family of SCP phosphatases seem to be more involved in regulating transcription through dephosphorylation of a different protein called the RE-1 silencing transcription factor (REST). REST is a major silencer of neuronal gene expression in non-neuronal cells which helps prevent development of improper neuronal phenotypes. Abnormally high protein levels of REST have been found in subsets of glioblastoma isolates which likely contributes to their oncogenesis and resistance of chemotherapeutics. SCP1 upregulates REST protein levels through dephosphorylating two degron sites that normally promote rapid turnover of REST, making it a potential drug target for glioblastomas in future studies. In Chapter 4, we show structurally how SCP1 recognizes these REST phosphorylation sites through complex x-ray crystallography. Data presented in this chapter reveal SCP1 specificity for each REST site and how SCP1 activity towards both of them promote REST gene silencing function.

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