Mitochondrial biogenesis requires the coordinate expression of the nuclear and mitochondrial genomes. The long term goal of this research is to understand the nature of the controlling signals in Saccharomyces cerevisiae that synchronize the production of mitochondrial proteins encoded in the separate compartments. The characterization of nuclear respiratory deficient pet mutants has identified several genes necessary for processing of mitochondrial precursor transcripts and translation of the resultant mRNAs on mitochondrial ribosomes. One group of pet mutants describes the nuclear gene CBP1, which encodes a 76,5000 dalton protein that is imported into the organelle and interacts with the 5' end of the cytochrome b precursor RNA. The CBP1 gene exhibits a complex pattern of polyA+ RNAs which are regulated in abundance with respect to carbon source.
The specific aims of this proposal are to study the interaction of CBP1 protein with precursor RNA for cytochrome b in vitro, to delineate the site of action of CBP1 in vivo, and to investigate the regulation of CBP1 transcription. The CBP1 protein will be purified and its interaction with T3 generated substrate RNAs will be studied in vitro. The protein will be obtained from commercial or laboratory yeast by a combination of size exclusion, ion exchange and affinity chromatography, or by fusion to the yeast pheromone alpha-factor signal sequence, or by in vitro translation of T3-generated mRNA. The activity of the protein will be monitored by a variety of RNA binding and scission assays. The site of action of GBP1 in vivo will be investigated via linker-scanner mutagenesis of the 5' end region of the cytochrome b. gene. The CBP1 gene produces two different polyA+ RNAs: a full length 2.3 kb mRNA and a 1.3 kb RNA. from the 5' half of the gene. On switching yeast from fermentative to respiratory growth conditions, the 2.3 kb RNA decreases with a concomitant increase in the 1.3 kb RNA. How and why the abundance of the RNAs is regulated with respect to carbon source will be investigated by mutational studies. CBP1 is closely flanked by the URF (unknown function) and NUC1 (mitochondrial nuclease) genes. A deletion in URF up-regulates CBP1 expression. The influence of URF and NUC1 expression on that of CBP1 will be studied via deletion and insertional mutagenesis. Changes in RNA levels and 5'/3 ends will be monitored by Northern and S1 analyses respectively.
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