The objective of the proposed research is to identify different subdomains of mtRNA polymerase that are involved in various polymerase functions (i.e., stability, catalytic activity, promoter recognition, interaction with the specificity factor, import into mitochondria, etc.). In particular, the role of amino acid(s) or amino acid sequence (s) in the function of polymerase will be evaluated. Temperature sensitive and deletion/substitution mutants of the mtRNA polymerase will be generated from a cloned mtRNA polymerase gene (RP041) using classical and recombinant methods. The activity of the altered polymerase will be tested in vivo and in vitro, after introducing the modified rpo41 gene into a haploid yeast carrying a nonfunctional chromosomal rpo41 gene by plasmid shuffle or mating and meiotic segregation. The specific alterations of polymerase function will be correlated with the particular amino acid or structural changes. This information in conjunction with previous in vitro transcription results will provide a better understanding of polymerase function in mitochondrial transcription. The work presented in this research proposal is relevant to muscle tissue function which is almost completely dependent upon the aerobic synthesis of ATP by mitochondria. Yeast, a unicellular facultative anaerobe, is used as a model system since this is the only convenient eukaryotic system for a genetic analysis of nuclear-mitochondrial interactions.
