Date of Award

Spring 1-1-2010

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

First Advisor

Dylan J. Taatjes

Second Advisor

Xuedong Liu

Third Advisor

Amy Palmer

Abstract

RNA polymerase II coordinates with a wide range of factors and catalyzes DNA transcription to synthesize mRNA. One critical step of transcription initiation is the interaction between the Mediator complex and the carboxy-terminal domain (CTD) of the largest subunit of human RNA polymerase II that consists of 52 repeats of the general consensus sequence YSPTSPS. This Mediator-Pol II CTD interaction is believed to be important in transcriptional regulation, RNA processing and chromatin remodeling. Therefore, elucidating the mechanism of this interaction is crucial for understanding gene expression and developing potential clinical applications. Our lab used a chemical crosslinking method in order to identify which Mediator subunit(s) interact with CTD. The results indicated that there might be two or three Mediator subunits bound to the Pol II CTD. Western blot experiments using antibodies specific for Mediator subunits revealed that Med1 and Med23 likely interact with CTD. In addition, in vitro binding assays show that Med1 binds to the first half of CTD (repeats 1-24), but not to the second half (repeats 25-52). The molecular details of the interaction need to be explored using mass spectrometry along with combined approaches of molecular biology and biochemistry.

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