The Regulatory Role of Phosphorylation of Mitochondrial Transcription Factor B2 (TFB2M)

Abstract

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial DNA (mtDNA) encodes thirteen protein subunits required for oxidative phosphorylation. The remaining mitochondrial proteome is encoded by the nuclear genome; which includes additional oxidative phosphorylation subunits, as well as the proteins necessary for mtDNA replication, expression and stability. Therefore, to respond to metabolic changes, mitochondrial gene expression must be coordinated with nuclear gene expression. While the control of nuclear gene expression is widely studied, there is a need to understand the regulation of mtDNA transcription. Our central hypothesis is that reversible protein post‐translational modifications of the mtDNA transcriptional machinery is a means to tune mtDNA transcription in response to changes in cellular metabolism. To address this gap in knowledge, this research focuses on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow for the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, threonine 313 and serine 197. To mimic the behavior of phosphorylation on these three sites, individual amino acids were mutated to glutamate, an amino acid that mimics the size and charge of the phosphoryl group. WT and phosphomimetic TFB2M were expressed in E. coli and purified. The purified proteins were analyzed for their ability to bind mtDNA through a pull‐down assay and fluorescence polarization was used to determine the binding dissociation constants for the light strand promoter. Interactions between TFB2M phosphomimics and POLRMT were also assessed. The data support the hypothesis that the phosphorylation at these sites may decrease the ability of TFB2M to bind to mtDNA. In vitro transcription assays were performed to determine the impact of these modifications on TFB2M’s role in transcription initiation. It is expected that these findings will lead to a more complete understanding of the dynamics and coordination of mitochondrial and nuclear gene transcription, as well as adding to the understanding of the importance of protein post‐translational modifications in regulating aspects of mitochondrial function.Support or Funding InformationThis material is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.