Genetic methods will be employed to elucidate the mechanism of upstream activation of transcription of the CYCl gene of Saccharomyces cerevisiae, and the coordination of CYCl activation with that of other cytochrome genes. Base substitution mutations of the CYCl upstream activation sites (UASs) will be generated as well as mutations in genes encoding trans-acting proteins which mediate the activation. Regulatory genes will be cloned, their sequence and regulation determined, and their encoded products expressed to high levels in E. coli and yeast. In vitro assays for binding of these proteins to the UASs will be developed and used to purify the proteins. The purified proteins will be studied biochemically and employed in an in vitro transcription system programmed by CYCl DNA. The molecular mechanism of upstream activation will be studied in vivo and in the purified system. The regulatory circuits governing coordinate control of nuclear and mitochondrial cytochrome genes will be deduced by an examination of the pleiotropy of mutations affecting CYCl trans-acting regulators. Whether genes encoding subunits of the mitochondrial RNA polymerase are a part of this circuit will be determined by molecular analysis after these genes are cloned.
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