Date Approved

10-2016

Document Type

Dissertation

Degree Name

Ph.D. in Cell and Molecular Biology

Department

Cell Biology

College

Graduate School of Biomedical Sciences

First Advisor

Dmitry Temiakov, PhD

Committee Member 1

William T. McAllister, PhD

Committee Member 2

Eric Moss, PhD

Committee Member 3

Natalia Shcherbik, PhD

Committee Member 4

Mikhail Anikin, PhD

Subject(s)

RNA, Mitochondrial, Gene Expression, Transcription, Genetic, Humans, Transcriptional Elongation Factors

Disciplines

Cell Biology | Genetic Processes | Genetics and Genomics | Laboratory and Basic Science Research | Medicine and Health Sciences | Microbiology | Molecular Genetics

Abstract

Coordinated replication and expression of mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mtDNA copy number can be regulated by the transcription machinery. Human mitochondrial RNA polymerase (mtRNAP) is a central enzyme involved in gene expression in mitochondria. It generates genome-size polycistronic transcripts and also makes replication primers at two origins of replication. MtRNAP is distantly related to phage T7 RNAP. While T7 RNAP is optimized to produce large amounts of transcripts to overcompete the bacterial RNAP, mtRNAP must coordinate RNA synthesis with processing and translation. We hypothesized that mtRNAP must be slower than T7 RNAP and measured elongation rates for these RNAPs. We found that mtRNAP is about 20 times slower than T7 RNAP. We also found that mtRNAP is inherently non-processive and cannot synthesize long transcripts. We proposed that low processivity and slow elongation rates of mtRNAP requires assistance of an additional elongation factor. We show that interaction of a recently identified human transcription elongation factor, TEFM, with mtRNAP dramatically increases processivity and elongation rates of the mitochondrial transcription machinery. Importantly, we found that TEFM prevents premature transcription termination and thus generation of replication primers by mtRNAP. Thus, TEFM serves as a component of a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. The switch likely allows avoiding the detrimental consequences of head-on collisions between replication and transcription machineries. Regulation of TEFM may explain how mtRNAP transcription rates and, as consequence, respiration and ATP production, can be increased in mitochondria without the need to replicate mtDNA, which has been observed during different developmental processes.

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