Mitochondria are abundant in the cells of such metabolically active tissues as brain, skeletal muscle, heart, kidney and liver. These organelles maintain and express a multi-copy genome, thereby generating proteins that are involved in oxidative phosphorylation. Human cells differ in the levels of mtDNA they contain. Somatic cells usually have ~1000-3000 genome copies but copy number can be as low as ~100 in spermatozoids and as high as 500,000 in oocytes. Changes in mtDNA copy number occur during normal developmental processes but have also been reported during various pathological processes and aging. Thus, decreased copy number has been observed during neurodegeneration, renal cell carcinoma, cardiomyopathy and breast cancer, while increased mtDNA copy number was shown to negatively affect male fertility. At present, the mechanisms responsible for regulation of mtDNA copy number are poorly understood, impeding our ability to use mitochondria as a therapeutic target. Mitochondria have a unique genetic system and at present, there is a gap in our understanding how mtDNA expression is regulated in cells. The replication machinery in human mitochondria includes DNA polymerase ?, TWEENKLE helicase, single-stranded DNA binding protein and RNA polymerase, which generates replication primers. Recent findings identified mitochondrial transcription elongation factor, TEFM as a major component of a molecular switch between replication and transcription in human mitochondria. The goal of this project is to determine how this switch is regulated in mitochondria of living cells.
The specific aims are as follows:
Aim 1. Determine the role of functional domains of TEFM in replication and transcription of mtDNA. Activities of TEFM will be investigated in biochemical assays that include transcription elongation and termination, as well as in nuclease activity assay using nucleic acid scaffolds of various topologies. The crystal structure of TEFM will be determined at atomic resolution to guide biochemical experiments designed to probe the functional importance of its domains.
Aim 2. Determine the molecular mechanism of TEFM action. The crystal structure of the anti-termination complex will be determined to elucidate the molecular mechanisms of TEFM action. Using protein-protein cross-linking and mass-spectrometry we will probe the organization of anti-termination complexes at different stages of transcription and replication.
Aim 3. Determine how the replication-transcription switch operates in human mitochondria. We will determine how changes in TEFM expression affect mtDNA copy number and mitochondrial transcription in cells during normal and stress conditions, and during cell differentiation. We will investigate how modulation of TEFM expression affects synthesis of the 7S DNA and how post-translational modifications of TEFM and mtRNAP contribute to the regulation of replication-transcription switch in cells.

Public Health Relevance

Changes in mitochondrial DNA copy number have been associated with neurodegeneration, cancer, and infertility. Studies of regulation of human mitochondrial DNA replication and transcription can be used to identify mechanisms or substances that impair or stimulate gene expression. This knowledge will help development of the treatments of human diseases that result from aberrant activity of replication and transcription machineries in mitochondria.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM118941-03
Application #
9827255
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Sledjeski, Darren D
Project Start
2017-07-15
Project End
2021-05-31
Budget Start
2018-09-01
Budget End
2019-05-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Hillen, Hauke S; Parshin, Andrey V; Agaronyan, Karen et al. (2017) Mechanism of Transcription Anti-termination in Human Mitochondria. Cell 171:1082-1093.e13