The goal of the proposed research is to gain an understanding of the steps of eukaryotic pre-tRNA and pre-rRNA maturation and the gene products that play a role in these processes. In all organisms initial transcripts can differ significantly from the mature RNAs that function in protein synthesis. Although there is a general outling of the steps necessary to generate mature tRNAs and rRNAs, few of the enzymes that catalyze nucleolytic, splicing and modification reactions have been characterized and even fewer of the genes coding and regulating these activities have been studied. Furthermore, it is known that pre-rRNA processing steps occur upon ribonuclear particles and it is possible that such particles participate in pre-tRNA processing. Thus, gene products other than those responsible for the catalytic activities are also likely to be required for processing reactions. We are utilizing genetic, molecular and biochemical approaches to answer four questions regarding the processing of pre-tRNA and pre-rRNAs in Saccharomyces cerevisiae: (1) What parts of pre- tRNAs are important to the processing machinery? We will study the in vivo consequences upon pre-tRNA processing of single mutations in a gene encoding tRNATyr; (2) What products are involved in pre-tRNA processing? We are attempting to identify, by mutation or by gene amplification, genes that affect the maturation of tRNA; (3) Is pre-tRNA processing associated with ancillary superstructural proteins? (4) What are the functions of the genes that previously had been shown to affect pre-tRNA and pre-rRNA processing? We have now identified 6 genes (RNA1, SRN1, LOS1, pC6, RRP1 and SRD1) that affect processing. Surprisingly, it does not appear that any of these code for known catalytic activities and therefore these genes may serve to identify products that were unanticipated from the biochemical pathways of the processing reactions. Our approach to study these genes is to clone the wild-type genes, to locate the gene products within yeast, to biochemically characterize these products, and to identify other genes with related functions.
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