Post‐Translational Modifications of Mitochondrial Proteins MRPL12 and POLRMT as a Potential Regulatory Mechanism for Mitochondrial DNA Transcription

Mitochondrial DNA (mtDNA) encodes 13 of the protein subunits of the oxidative phosphorylation pathway, the pathway that produces the majority of ATP in eukaryotic cells. Mitochondrial dysfunction is associated with neurodegenerative disorders, cancer, inherited mitochondrial diseases, and other pathologies, making mitochondrial biochemistry an important area of investigation. However, there lacks an understanding of key elements of this biochemistry, including the mechanisms that regulate mtDNA transcription. mtDNA is known to be complexed to various proteins in structures known as nucleoids. Some nucleoid proteins are key members of the mtDNA transcription machinery; these include the mitochondrial RNA polymerase (POLRMT) and mitochondrial ribosomal protein L12 (MRPL12), which maintains POLRMT stability and promotes mtDNA transcription. We hypothesize that reversible post‐translational modifications (PTMs), such as phosphorylation of threonine, serine, or tyrosine residues or acetylation of lysine residues within these and other nucleoid proteins, may play a role in regulating mtDNA transcription. The objective of our study is to determine the effects of MRPL12 and POLRMT post‐translational modifications on the activities of these proteins in the context of mtDNA transcription in order to assess the roles of such modifications in regulating this process. To test this, we performed mutagenesis PCR on MRPL12 and POLRMT genes within bacterial expression vectors to replace the sequence encoding an amino acid at a PTM site with a sequence encoding a modified or unmodified amino acid mimic (T, S, Y → E/A, phosphorylated/dephosphorylated mimic; K → Q/R, acetylated/deacetylated mimic). We optimized bacterial expression and protein purification conditions and purified wild‐type and mutant MRPL12 proteins, as well as wild‐type POLRMT. We performed preliminary in vitro transcription assays to assess POLRMT functionality and found that our protein successfully transcribed short mtDNA templates. Going forward, we will perform protein‐protein binding assays to determine the effects of MRPL12 modifications on MRPL12's ability to bind POLRMT, as well as POLRMT mutagenesis and more in vitro transcription assays to measure the effects of MRPL12 and POLRMT modifications on mtDNA transcription. Support or Funding Information This research is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845, with additional funding from the Arnold and Mabel Beckman Foundation Beckman Scholars Award and the ASBMB Undergraduate Research award. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .