Mitochondrial biogenesis requires the coordinate expression of the nuclear and mitochondrial genomes. The long term goal is to understand the nature of the controlling signals in Saccharomyces cerevisiae that synchronize the production of mitochondrial constituents encoded in the separate compartments. The characterization of nuclear respiratory deficient strains of yeast, pet mutants, has identified several nuclear genes necessary for proper processing of mitochondrial transcripts and translation of the mRNAs.
The aim of this proposal is to investigate the role of two nuclearly encoded mitochondrial RNA processing enzymes in regulating the synthesis of mitochondrially encoded proteins. A nuclear gene product, initially identified by pet mutations, is specifically responsible for conferring a stable 5' terminus on the mitochondrial mRNA for cytochrome b, the only mitochondrially encoded subunit of coenzyme Q - cytochrome c reductase. This CBP1, (cytochrome b processing), protein will be further purified by column chromatography, and its action will be studied in vitro. RNAs produced in an SP6 bacteriophage promoter-transcription system will be used as substrates. The sequence and structural requirements of the RNA substrate for recognition by the protein will be investigated by characterizing mitochondrial suppressors that alleviate cbp1 mutations, and by testing altered RNA substrates in the in vitro assay. Another protein will be isolated that is responsible for cleaving mitochondrial multigenic primary transcripts into unigene segments, thereby conferring mature 3' termini on several mitochondrial mRNAs. Purification of this endonuclease will be accomplished by one of two approaches. The protein will be purified by conventional chromatographic methods, employing an in vitro functional assay with artificially SP6-generated RNA substrates. Using antibodies raised to this protein, the nuclear gene encoding the enzyme will be selected from a cDNA expression library. Alternatively, an indirect approach will be used, involving characterization of pet mutants defective in this activity, isolation of the gene by transformation with cloned wild-type yeast DNA, production of an antibody to an overexpressed E. coli trp E-3'processing gene fusion product, and immuno-detection of the protein throughout a chromatographic procedure. To ascertain whether the genes encoding these proteins are under a higher order regulatory system that coordinates several nuclearly encoded mitochondrial components simultaneously, changes in the levels of mRNAs produced for the specific 5'-end and general 3'-end endonucleases will be monitored when wild-type yeast is switched from glucose repression conditions to growth on a non-fermentable carbon source.
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