# Molecular Mechanisms of Mitochondrial Transcription and Replication

> **NIH NIH R35** · THOMAS JEFFERSON UNIVERSITY · 2022 · $533,520

## Abstract

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PROJECT SUMMARY
Defects in mtDNA replication and expression result in myopathies, hearing and vision loss, and other
devastating disorders. The key enzyme involved in mtDNA expression, mitochondrial RNA polymerase
(mtRNAP) belongs to a family of single-subunit RNAPs that is distinct from the multi-subunit cellular RNAPs.
Human mtDNA contains only two promoters that share limited sequence homology; the molecular basis for
recognition of these promoters remains unclear. We will use the recently obtained structures of the initiation
complex to guide biochemical experiments and probe the function of mtRNAP domains involved in promoter
binding, recognition, and melting. Our studies will determine the role the initiation factors play in the
mechanisms of promoter recognition. We will obtain the high-resolution structures of the pre-initiation and
initially transcribing initiation complexes. In the absence of transcription initiation factors mtRNAP is capable to
initiate transcription on a hairpin promoter at the origin of replication OriL and thus generates replication
primers. We will elucidate how mtRNAP recognizes and binds this unusual promoter and what factors affect
this process. The structure of OriL-IC will be determined. Studies of the structure and function of the mtRNAP
and molecular mechanisms of transcription and replication are important for understanding the regulation of
mitochondrial genome expression. This, in turn, will determine our ability to influence various mitochondrial
functions and as a consequence, to treat mitochondria-associated diseases.
Human cells differ in the levels of mtDNA they contain. Changes in mtDNA copy number occur during normal
developmental processes but have also been reported during various pathological processes and aging. At
present, the mechanisms responsible for regulation of mtDNA copy number are poorly understood, impeding
our ability to use mitochondria as a therapeutic target. The replication machinery in human mitochondria
includes DNA polymerase gamma, TWINKLE helicase, single-stranded DNA binding protein and mtRNAP,
which generates replication primers. Our recent studies identified mitochondrial transcription elongation factor,
TEFM, as a major component of a molecular switch between replication and transcription in mitochondria that
would allow the corresponding machineries to avoid the detrimental consequences of head-on collisions. How
this switch is regulated in cells in response to different conditions is not understood. This research with
determine molecular mechanism of TEFM action by biochemical and structural approaches and how changes
in TEFM expression affect mtDNA copy number and mitochondrial transcription in cells during normal and
stress conditions, and during cell differentiation. To elucidate the underlying mechanisms that control the set
level of mtDNA, the interplay between the TEFM-dependent switch and the switch that operates at the D-loop
region of mtDNA and regulates uti...

## Key facts

- **NIH application ID:** 10406992
- **Project number:** 5R35GM131832-04
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Dmitry Temiakov
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $533,520
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10406992

## Citation

> US National Institutes of Health, RePORTER application 10406992, Molecular Mechanisms of Mitochondrial Transcription and Replication (5R35GM131832-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10406992. Licensed CC0.

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