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Mechanism and regulation of yeast and human mitochondrial DNA transcription

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TitleInfo
Title
Mechanism and regulation of yeast and human mitochondrial DNA transcription
Name (type = personal)
NamePart (type = family)
Basu
NamePart (type = given)
Urmimala
DisplayForm
Urmimala Basu
Role
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author
Name (type = personal)
NamePart (type = family)
Hampsey
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Michael
DisplayForm
Michael Hampsey
Affiliation
Advisory Committee
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chair
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
School of Graduate Studies
Role
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school
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Text
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theses
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2019
DateOther (qualifier = exact); (type = degree)
2019-05
Language
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English
Abstract (type = abstract)
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.
Subject (authority = local)
Topic
Transcription
Subject (authority = RUETD)
Topic
Biochemistry
Subject (authority = LCSH)
Topic
Genetic transcription
Subject (authority = LCSH)
Topic
Yeast fungi -- Genetics
Subject (authority = LCSH)
Topic
Mitochondrial DNA -- Genetics
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Rutgers University Electronic Theses and Dissertations
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ETD_9718
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application/pdf
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text/xml
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1 online resource (xiv, 131 pages) : illustrations
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
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School of Graduate Studies Electronic Theses and Dissertations
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rucore10001600001
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Identifier (type = doi)
doi:10.7282/t3-rfej-8c15
Genre (authority = ExL-Esploro)
ETD doctoral
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The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Basu
GivenName
Urmimala
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Copyright Holder
RightsEvent
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Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2019-04-08 15:37:08
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Urmimala Basu
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Affiliation
Rutgers University. School of Graduate Studies
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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Embargo
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2019-05-31
DateTime (encoding = w3cdtf); (qualifier = exact); (point = end)
2021-05-30
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after May 30th, 2021.
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Copyright protected
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Open
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2019-04-15T11:35:02
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2019-04-15T11:35:02
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