Mitochondria are essential organelles that provide a bulk of cellular energy and contribute to many other important functions. Mitochondria contain their own DNA (mtDNA) which encodes a subset of genes necessary for the function of the organelle. These genes are expressed by mitochondria-specific transcription and translation machineries. Dysregulation of mitochondrial gene expression has been increasingly recognized as an important contributor to aging and to human diseases such as Parkinson's disease, hypertension, diabetes, and cancer. Notably, while major mtDNA regulatory elements and basal machinery required for human mitochondrial DNA expression has been identified, many important questions about the mechanism and regulation of transcription in mammalian mitochondria remain. Here, we propose a series of biochemical and genetic experiments to investigate for the first time the dynamics of human mitochondrial transcription complexes and their interaction with mitochondrial translation machinery. In Aim 1 of this proposal, transcription factors interacting with the three mtDNA promoters will be identified and analyzed. Interaction of basal transcription factors with mtDNA will also be investigated via chromatin immunoprecipitation approach. In Aim 2, connections between transcription and translation machineries in human mitochondria will be addressed. Affinity purification of transcription complexes followed by Western blotting and mass-spectrometry will be used to study transcription-translation coupling in mitochondria. Finally, in Aim 3, we will investigate the function of MRPL12, a unique mitochondrial ribosomal protein that interacts with mitochondrial RNA polymerase and modulates transcription of mitochondrial genes. Effects of MRPL12 overexpression on mitochondrial biogenesis and function will be analyzed. Functional significance of MRPL12 interaction with transcription machinery will also be examined. Completion of the experiments described in this proposal will provide fundamental new insight into mitochondrial transcription and will greatly increase our understanding of basic mitochondrial biology. Moreover, this study will have a large impact on the search for potential therapeutic strategies based on modulating mitochondrial gene expression and mitochondrial DNA replication. ) PUBLIC HEALTH RELEVANCE: Mitochondria are essential organelles that contain their own DNA (mtDNA), dysregulation of which is implicated in human disease and aging. While modulation of mtDNA expression is recognized as a potential therapy for mitochondrial pathology, this approach is limited by our incomplete knowledge of how this process is regulated. The detailed analysis of human mitochondrial transcription complexes in this proposal will greatly increase our understanding of mitochondrial gene expression and bring us closer to this goal.