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The Effect of Ssu72-Mediated RNA Polymerase II CTD Dephosphorylation on Progression through the Transcription Cycle

by
Jesus D. Rosado-Lugo
B.S., Pontifical Catholic University of
Puerto Rico - 2005


Thesis Advisor: Michael Hampsey, Ph.D.
Graduate Program in Biochemistry

Biochemistry Department Conference Room
6th floor, RWJMS Research Tower
Piscataway

Tuesday, September 4, 2012
1:00 p.m.


Abstract

In eukaryotes RNA Polymerase II (RNAPII) is in charge of transcribing protein-coding genes (mRNA genes), some small nuclear RNA (snRNA) genes as well as many non-coding RNAs (ncRNAs). Transcription by RNAPII occurs in distinct stages that include assembly of the pre-initiation complex (PIC), promoter melting, initiation, promoter clearance, elongation and termination. During the different stages of the transcription cycle additional factors need to be recruited to RNAPII like the mRNA capping enzymes, elongation factors, splicing factors, 3-end processing factors and termination factors. Recruitment of these factors to RNAPII is coordinated by the dynamic phosphorylation of a C-terminal domain (CTD) in the largest subunit of RNAPII (Rpb1). The CTD is composed of multiple heptapeptide repeats (YS2PTS5PS7) that undergo reversible phosphorylation on all serine residues at different stages of the transcription cycle. In yeast, the Ssu72 phosphatase is a CTD phosphatase with specificity against Ser5-P and Ser7-P that is essential for cell viability. The role of Ssu72 in the transcription cycle is especially enigmatic since it physically associates with TFIIB, the Rpb2 subunit of RNAPII, the Taf2 subunit of TFIID, and the Kin28 subunit of TFIIH. These interactions implicate Ssu72 in initiation. Yet Ssu72 is an integral component of the 3-end cleavage-polyadenylation factor (CPF) complex where it plays a role in 3-end processing and termination. At the outset of this work, it was not known where in the transcription cycle Ssu72 acted, or if its phosphatase activity regulated RNAPII progression through the transcription cycle. Also, the recent discovery of another phosphatase called Rtr1, with specificity towards Ser5-P as well, raises the questions of whether or not they have redundant activities. In this thesis, I will answer the following questions regarding the phosphatase activity of Ssu72 and its relation to Rtr1. 1-Does the phosphatase activity of Ssu72 act during the transcription cycle? 2-At what stage of the transcription cycle does Ssu72 act? 3-Will loss of function of Ssu72 affect RNAPII progression trough the transcription cycle? 4- Do Ssu72 and Rtr1 have redundant phosphatase activities towards the CTD?


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