Mitochondria have their own genome, and many key proteins of oxidative phosphorylation (OXPHOS) are encoded in this genome. Mitochondrial RNA polymerase (POLRMT) is responsible for mitochondrial genome transcription and its transcription is regulated by several transcription factors. Myocyte-specific enhancer factor 2D (MEF2D) belongs to the MEF2 family that enhances myocyte-specific gene expression in the nuclear genome by interacting with other transcription regulation factors. It was reported that MEF2D enhances the light strand of mitochondrial genome transcription by binding to a specific sequence in the NADH dehydrogenase 6 (ND6) gene in the light strand. By overexpression of dominant negative MEF2D, mice were sensitized to the toxicant commonly used to trigger Parkinson’s disease...[ Read more ]

Mitochondria have their own genome, and many key proteins of oxidative phosphorylation (OXPHOS) are encoded in this genome. Mitochondrial RNA polymerase (POLRMT) is responsible for mitochondrial genome transcription and its transcription is regulated by several transcription factors. Myocyte-specific enhancer factor 2D (MEF2D) belongs to the MEF2 family that enhances myocyte-specific gene expression in the nuclear genome by interacting with other transcription regulation factors. It was reported that MEF2D enhances the light strand of mitochondrial genome transcription by binding to a specific sequence in the NADH dehydrogenase 6 (ND6) gene in the light strand. By overexpression of dominant negative MEF2D, mice were sensitized to the toxicant commonly used to trigger Parkinson’s disease symptoms, therefore ND6 regulation by MEF2D may be clinically interesting. However, the detailed transcription regulation mechanism by MEF2D in mitochondrial genomic DNA is not well understood. I used bulk transcription assay, precision nuclear run-on sequencing (Pro-Seq) pull-down assay to investigate the effect of MEF2D on POLRMT transcription. Furthermore, I attempted to optimize the high-resolution optical tweezers for the single-molecule transcription assay to quantify the effect of MEF2D. The tweezers failed to reach the required efficiency in performing single-molecule POLMRT transcription assay. I found some operation limitations of the high-resolution optical tweezers in performing single-molecule POLRMT transcription assay. The refitted high-resolution optical tweezers, however, are still suitable for other single-molecule experiments. The optimization and experience obtained from attempting the POLRMT transcription assay may benefit future single-molecule experiments using the refitted high-resolution optical tweezers.

Key words: Mitochondrial RNA Polymerase, MEF2D, Transcription Elongation, Optical Tweezers, ND6, Microfluidics