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Mechanistic studies of transcription initiation by S. cerevisiae mitochondrial RNA polymerase

by
Aishwarya Deshpande
Institute of Bioinformatics & Biotechnology, University of Pune - 2008

Thesis Advisors: Smita S. Patel, Ph.D.
Graduate Program in Biochemistry

School of Public Health Building
2nd floor Conference Room 258
Piscataway

Thursday, July 17, 2014
2:00 p.m.


Abstract

Mitochondria are the major suppliers of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mt and aging related disorders; however very little is known about the basic mechanisms of processes that regulate mitochondrial gene expression. This dissertation examines two aspects of transcription initiation by the mitochondrial RNA polymerase, using Saccharomyces cerevisiae (yeast) as the model system.
The yeast mitochondrial transcription machinery consists of two nuclear-encoded proteins, the core RNA polymerase subunit Rpo41 and the accessory transcription factor Mtf1. The two proteins form a complex on promoters containing the 5-(-8)ATATAAGTA(+1) consensus sequence and initiate specific transcription from +1+2 AA, AG or AT sequence with varying efficiencies that is important for regulating expression of RNAs and proteins involved in ATP production. This thesis investigates the biochemical basis of the varying efficiencies of mitochondrial promoters using 2-aminopurine fluorescence studies that monitor promoter melting and kinetics of 2-mer synthesis that monitor the efficiency of transcription initiation. The studies were carried out with natural promoters and minimal promoters with systematic changes in the +1+2 base pairs to matched and mismatched pairs. My results show that Rpo41-Mtf1 can melt the upstream -4 to -1 region in all promoters irrespective of the +1+2 sequence, but it initiates efficiently only on promoters with +1+2 AA. This is because Rpo41-Mtf1 has specific interactions with the +1+2 non-template bases that result in efficient melting of the downstream +1+2 region and formation of a stable pre-transcribing complex. On the other hand, inefficient +1+2 melting of AG and AT promoters increases the amount of initial NTPs needed to form a stable pre-transcribing complex by 10-20 fold relative to the AA promoter or singly mispaired promoters, which explains their poor initiation efficiencies.
Rpo41 shares with other mtRNAPs a conserved C-terminal catalytic domain (~800 aa) that is highly homologous to the catalytic domain of the single-subunit bacteriophage T3/T7 RNAPs. However, the N-terminal domain (NTD) (~400 aa) of Rpo41 is less conserved and absent in phage RNAPs. In order to understand the role of this unique NTD in mitochondrial transcription, I studied a series of truncated Rpo41 proteins with systematic NTD deletions. My studies show that NTD interacts with Mtf1 and this interaction is important for efficient initiation, but on pre-melted promoters, the presence of NTD inhibits transition from initiation to elongation. Using cross-linking assays, I show that Rpo41 interacts partially via its NTD with Mtf1 to form an initial complex in 1:1 stoichiometry, even in the absence of promoter. The pre-melted promoters from -4 to +2 fail to transition from initiation to elongation efficiently in the presence of Mtf1, but deletion of the NTD restores efficient transcription. In addition to providing basic mechanistic insights, the efficient transcription by the NTD mutants has potential applications in developing commercial tools for custom RNA synthesis.


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