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Transcription by S.cerevisiae mitochondrial RNA polymerase

by
Maria Savkina
Bachelor of techinics and technology, 2001
Moscow State Academy of Fine Applied Chemistry
Master of techinics and technology, 2003
Moscow State Academy of Fine Applied Chemistry

Thesis Advisor: William T. McAllister, Ph.D.
Cell and Molecular Biology Program

Science Center, Room 290

Monday, May 11, 2009
1 pm


Abstract

Yeast mitochondrial core RNA polymerase (mtRNAP) is a single subunit enzyme (Rpo41p) that shares significant sequence homology with T7 RNAP. However, unlike T7 RNAP, Rpo41p requires an additional factor (Mtf1p) for transcription initiation. It was initially proposed that Mtf1p is a specificity factor that is required for promoter recognition, however subsequent studies suggested that Mtf1p is a transcription activator that is needed for promoter melting, and that Rpo41p has the intrinsic ability of promoter recognition.

We studied the roles of Rpo41p and Mtf1p in transcription initiation in a number of ways. First, using synthetic promoter templates we determined the boundaries and topology of the melted region that are required for Rpo41p to initiate in the absence of Mtf1p. Second, using DNA:protein photo cross-linking methods we found that the non-template (NT) strand of the promoter cross-links predominately to Rpo41p and that the template (T) strand interacts with both Rpo41p and Mtf1p. We identified a C terminal domain (amino acids 320 -341) in Mtf1p that interacts with the -3 residue in the T strand.

To examine the properties of mtRNAP elongation complexes (EC) we assembled functional transcription complexes on nucleic acid scaffolds using an approach that was previously explored to characterize T7 RNAP ECs and explored the contribution of different components of the nucleic acid scaffold to the stability of the mtRNAP EC.

The mechanism by which mtRNAP recognizes its promoters is not known. In the related T7 RNAP system, promoter recognition involves a “specificity loop” that interacts with the upstream region of the promoter. Sequence homology between T7 RNAP and Rpo41p suggests that an analog of the specificity loop may be present in Rpo41p. To determine whether this element is important during transcription initiation by Rpo41p, we substituted amino acids in this region and examined the effects of these mutations on promoter recognition. We found that the region encompassing residues 1124-1151 is important for transcription initiation and may be involved in promoter recognition.


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