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Structural and biochemical characterization of S.cerevisiae mitochondrial RNA polymerase

by
Swaroopa Paratkar
B.S., Pharmaceutical Sciences - 2005
University of Mumbai


Thesis Advisor: Smita S. Patel, Ph.D.
Graduate Program in Biochemistry & Molecular Biology

School of Public Health building
2nd floor Conference Room
Piscataway

Friday, September 10, 2010
1:30 p.m.


Abstract

Mitochondria are cellular organelles responsible for ATP production in the eukaryotes. The proteins that replicate and transcribe the mitochondrial genome are made in the nucleus and transported into the mitochondria. The S.cerevisiae mitochondrial (mt) genome is transcribed by a core RNA polymerase, Rpo41 (150 kDa) and a specificity factor, Mtf1 (42kDa) that form a complex termed mt RNAP. The C-terminal two-thirds of Rpo41 is highly homologous to the bacteriophage T7/T3 RNAP. Rpo41 has a unique N-terminal domain (~400aa) whose role in transcription is not known. Rpo41 by itself does not recognize and melt the mt promoter or initiate RNA synthesis unless the promoter is pre-melted around the transcription site. When Mtf1 and Rpo41 are present together, the complex melts the promoter from −4 to +2 without requiring initiating NTPs. Based on these results, it has been suggested that Rpo41 lacks the mechanism for melting/ stabilizing the open promoter and relies on Mtf1 for promoter-specific binding, melting, and stabilizing of the melted promoter. Once the promoter is melted and the transcription start site exposed, mt RNAP initiates RNA synthesis and undergoes rounds of abortive initiation. Abortive initiation is a process in which RNA transcripts, often shorter than 10 bases are released from the RNA polymerase complex. As a consequence, only a fraction of the transcription initiation events lead to the next stage in transcription.

My dissertation focuses on understanding the role of Mtf1 and the unique N-terminal domain of Rpo41 in the initial steps of transcription including promoter specific DNA melting and abortive RNA synthesis. In the first part of my thesis, I reconstituted the S. cerevisiae mitochondrial transcription machinery in vitro using bacterially expressed proteins. In the second part, I have discovered several aspects of the mechanism by which Mtf1 facilitates promoter melting in the mtRNAP complex. By using protein-DNA photochemical crosslinking of mitochondrial transcription complex, I was able to show that the specificity factor Mtf1 is in close proximity to the entire promoter region and crosslinks efficiently with the unwound non-template strand. These studies indicated that Mtf1 directly binds to mitochondrial promoter and this interaction may stabilize the resulting ‘open’ complex. In the third part, I investigated the role of the N-terminal domain of Rpo41 in transcription initiation and elongation. By systematically deleting the N-terminal region of Rpo41, I studied the truncated proteins for their ability to melt the promoter DNA and catalyze sequence specific transcription. Deletions within the N-terminal domain from 1-380 caused reduced accumulation of short abortive transcripts relative to the full length protein. Deletion of the N-terminal region beyond amino acid 270 resulted in defective promoter melting. These studies suggest that the N-terminal domain of Rpo41 may aid in promoter opening by helping to stabilize the open complex. At the same time, the N-terminal domain appears to inhibit the transition from initiation to elongation; thus, its removal results in efficient runoff RNA synthesis with little abortive synthesis.

The insights gained from these studies can be extended to the highly homologous plant and human mitochondrial transcription and provide an intriguing mechanistic link to other multi-subunit RNA polymerases.


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