This proposal outlines molecular genetic and biochemical experiments designed to increase our understanding of the structure and function of the mitochondrial RNA polymerase from Saccharomyces cerevisiae. In particular, the subunit structure of the multicomponent enzyme will be determined and the possibility that multiple functional forms exist in the cell will be tested. We will characterize the interactions of the multiple components of the enzyme and study the interaction of the RNA polymerase(s) with promoter containing DNA. The expression of genes in both the nucleus and the mitochondria must be coordinately regulated to produce stoichiometric levels of mitochondrial precursors. In general, gene products from both compartments are repressed by growth on glucose. We have shown that the mitochondrial RNA polymerase is encoded by nuclear genes and is subject to glucose repression. To test the possibility that the level of available RNA polymerase may control mitochondrial gene transcription. We will study the expression of the nuclear genes which encode the RNA polymerase. One gene, encoding the catalytic core of the RNA polymerase, has been identified (RPO41). We will isolate the nuclear gene(s) for the additional specificity factor(s) which interacts with the core and confers functional promoter recognition. The cloned genes, and mutant yeast cells lacking the gene products, will be used to test the function and expression of the RNA polymerase subunits. Another possible level of co-ordinate control derives from our observations that many nuclear gene """"""""TATA"""""""" boxes (part of the RNA polymerase II promoter) are functional promoters for the mitochondrial RNA polymerase in vitro. We will test the possibility that a form of the polymerase specificity factor may interact with the nuclear transcription apparatus. We will also characterize a class of yeast genes (IMP, Independent of Mitochondrial Phenotype) which communicate the lact of functional mitochondria to a subset of nuclear genes. The IMP genes will be clonally isolated using a simple complementation assay. This analysis will begin to reveal the complex regulation involved in the maintenance of mitochondria in eukaryotic cells.
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