In vivo analysis of the 5' untranslated leader sequence of mitochondrial cytochrome b transcripts in Saccharomyces cerevisiae.
Committee ChairDieckmann, Carol L.
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PublisherThe University of Arizona.
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AbstractIn the yeast Saccharomyces cerevisiae the production of respiratory competent mitochondria requires the activity of several hundred proteins, the vast majority of which are encoded by nuclear genes and imported into mitochondria. A subset of these nuclear gene products are required for the expression of individual mitochondrial genes. Expression of the mitochondrial gene cob, encoding cytochrome b, requires the activity of at least seven nuclear gene products, including CBP1, CBS1 and CBS2. CBP1 is required for the stability of cob transcripts, while CBS1 and CBS2 are required for translation of cob mRNA. Previous analyses of mitochondrial rearrangements that suppress cbp1, cbs1, or cbs2 mutations have led to the hypothesis that CBP1, CBS1 and CBS2 interact with the 5' untranslated leader (UTR) of cob transcripts. To further define cob sequence important for stability or translation of cob transcripts, the expression of mutant cob genes, in which portions of the cob coding sequence or 5' UTR have been deleted, has been analyzed in vivo. Quantitation of steady-state levels of cob transcripts in wild-type CBP1 and mutant cbp1 strains carrying deletion mitochondrial genomes leads to the conclusion that a 63 nucleotide sequence is sufficient for the CBP1-dependent stability of cob transcripts. This sequence encompasses the site of cleavage that produces the mature 5' end of cob mRNA from precursor transcripts. Determination of the 5' ends of mature cob mRNAs in the deletion strains indicated that this sequence is also sufficient for correct positioning of the cleavage. The data suggest that the cleavage is CBP1-dependent and occurs at a specific distance 5' of a recognition site located within the defined 63 nucleotides. In addition, the data suggest that the stability of the mature cob mRNA produced by the cleavage is dependent on sequence at its 5' end. Lastly, an analysis of the structure of a high molecular weight cob transcript present in a respiratory incompetent deletion strain leads to the hypothesis that 5' UTR sequence between -200 and -4 is required for interaction with factors, such as CBS1 or CBS2, required for translation of cob transcripts.
Degree ProgramMolecular and Cellular Biology