The yeast mitochondrial genome is transcribed by a nuclear encoded RNA polymerase composed of a catalytic core related to bacteriophage enzymes and a promoter recognition factor related to bacterial sigma factors. We propose to analyze the interaction of this enzyme with its simple promoter. These experiments will use recombinant forms of the polymerase subunits and a collection of point mutations in the promoter to determine if sequence recognition is dependent on one or both subunits. We will also test other mitochondrial proteins to determine if additional factors affect promoter utilization. The genes encoding the subunits of the RNA polymerase will be used to identify homologues from other fungal species. Comparisons to these sequences and to genes encoding the bacteriophage and bacterial relatives of the subunits will guide our efforts to understand function by creating mutations in the mitochondrial polymerase genes. Yeast mitochondrial gene expression is regulated in response to carbon source. We will characterize this response by measuring changes in mitochondrial DNA copy number, RNA abundance, synthesis rates and stability. We will analyze the regulation of the nuclear genes encoding the polymerase subunits and test the involvement of nuclear genes known to control the process of glucose repression. We will use biochemical and genetic techniques to identify additional factors that may be important for regulation. These studies will aid our understanding of the coordinate expression of the nuclear and mitochondrial genomes and provide a model for mitochondrial gene expression in multicellular eukaryotes.
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