Date of Award

Spring 1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

James A. Goodrich

Second Advisor

Jennifer F. Kugel

Third Advisor

Natalie G. Ahn

Fourth Advisor

Robert Kuchta

Fifth Advisor

William M. Old

Abstract

Upon cell stress, the production of mRNA by RNA polymerase II (Pol II) is downregulated. In mouse cells B2 RNA, a ~180 nt non-coding RNA transcribed from retrotransposon elements, binds Pol II and represses transcription in response to heat shock. The studies detailed in this thesis examine the ncRNA binding site on Pol II, and the sequence determinants of B2 RNA that mediate repression of transcription in vitro. To identify the surface on Pol II to which B2 RNA binds, I developed a reversible crosslinking MS/MS assay coupled to a label free quantitation method. The crosslinking assay identified 36 Pol II peptides that crosslinked to B2 RNA(81-130), a region of B2 RNA that is fully functional. The label free quantitation revealed four of these peptides accounted for 92% of the total intensity. I mapped these highly enriched peptides onto a conserved yeast crystal structure and found the peptides are not only located in the DNA binding cleft and active site region of Pol II, but the peptides themselves also play key functional roles in the transcription reaction. To identify the sequence determinants of B2 RNA that allow it to repress transcription in vitro, I undertook a mutational strategy. Guided by an experimentally determined secondary structural model, I made a series of deletions from the 5' and 3' ends of B2 RNA, which revealed four different regions that function in repression of transcription in vitro. Deleting each of these regions showed that the unstructured region, 99-113, had the greatest effect on transcriptional repression in vitro. Moreover, deleting two unstructured regions, 99-113 and 153-178, from full length B2 RNA resulted in an RNA that no longer repressed transcription in vitro. Electrophoretic mobility shift assays (EMSAs) revealed that removing these transcriptionally repressive regions did not lead to a loss of Pol II binding by the ncRNAs. Hence, B2 RNA contains separate regions for binding Pol II and repressing transcription.

Included in

Biochemistry Commons

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