Mechanism and regulation of yeast and human mitochondrial DNA transcription
Creators: name: Basu, Urmimala
Publisher: name: No Publisher Supplied
Year: 2019

description: Mitochondria are autonomous double-membrane-bound organelles in eukaryotic cells. Their most important function is to synthesize ATP to meet the energy needs of the organism through oxidative phosphorylation. This cardinal role in energy production makes mitochondria a key player in metabolic, degenerative, and age-related diseases. Dysregulations of mitochondrial energy production in humans affect all organs, but the high energy demanding organs of the body like the heart, brain, kidneys, etc., are the primary targets of mitochondrial malfunctions. Mitochondria contain their own genome that is replicated and transcribed by enzymes distinct from the nuclear enzymes. Interestingly, the mitochondrial DNA (mtDNA) replication and transcription enzymes are homologous to bacteriophage T7 encoded enzymes. Thus, yeast and human mtDNA are transcribed by phage T7-like single-subunit RNA polymerases (RNAP) called Rpo41 and POLRMT, respectively. They are structurally homologous to T7 RNAP, but both yeast and human RNAPs require transcription factors to initiate transcription which includes: Mtf1 (in yeast) and mitochondrial transcription factor A /TFAM and mitochondrial transcription factor B2 /TFB2M (in humans). Reliance of the mtRNAP on transcription factors, results in regulation of gene expression that is unprecedented in homologous phage T7. Transcription initiation is a crucial step where gene expression is regulated. However, mtDNA transcription initiation, elongation, and termination mechanisms are understudied. The overarching goals of my graduate research were to investigate the mechanisms of regulation of transcription initiation through biochemical and biophysical characterization.In the first part of the thesis, I have showed that yeast and human mtRNAPs can initiate transcription with NAD+/NADH, which results in production of capped RNA transcripts in the mitochondria. Capping with NAD+/NADH is a new discovery brought to light in the last decade. My research has showed that initiation with NAD+ or NADH is up to 40% as efficient as initiation with ATP for S. cerevisiae mtRNAP and up to 60% as efficient as initiation with ATP for human mtRNAP. Similarly, direct quantitation of NAD+- and NADH-capped RNA in vivo showed up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts and up to ~10% NAD+ capping of human mitochondrial transcripts. Furthermore, both S. cerevisiae mtRNAP and human mtRNAP can cap RNA with NAD+ and NADH more efficiently than bacterial RNAP and eukaryotic nuclear RNAP II. The capping efficiency is higher with promoter derivatives having R:Y at position -1 than with promoter derivatives having Y:R. The implications of alternatively capping mitochondrial RNAs are not known; however, capping can affect RNA stability, processing, and global gene expression. Since intracellular NAD+ and NADH levels dictate the efficiency of capping, we propose that mtRNAPs use NAD+/NADH capping as both a sensor and actuator in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly. In the second part of the thesis, I studied the role of transcription initiation factors, Mtf1 and TFB2M, in transcription initiation. Specifically, I focused on understanding the function of the C-terminal region (C-tail) of Mtf1 and TFB2M. I engineered and purified recombinant proteins with deletions in the C-tail of S. cerevisiae Mtf1 and human TFB2M and investigated the effect of C-tail deletion on the various steps in transcription initiation. Using 2-aminopurine fluorescence-based studies, I have showed that the C-tail of Mtf1 is not necessary for promoter melting but the C-tail of TFB2M is essential for promoter melting. Nevertheless, deletion of the C-tail in both systems decreases the affinity for the initiating nucleotide, which indicated that the C-tail is critical for aligning the template strand in the active site. The biochemical phenotypes of C-tail deletion in Mtf1 resemble those of the sigma factor 3.2 region deletion in bacterial RNAP and B-reader loop in the RNA Pol II system. Thus, I have identified the mechanism of template alignment in mtRNAPs that is needed generally to initiate transcription from a specific site on the double-stranded DNA.