The proposed work is the continuation of a multifaceted project whose long range goal is an understanding of the mechanisms that regulate RNA stability as a key element of genetic control. The studies proposed for the next project period are intended to provide fundamental information about certain of the genes, enzymes, and substrate properties that govern RNA degradation. They will utilize a combination of genetic and biochemical approaches and will employ RNAI, an antisense repressor of the replication of ColEl-type plasmids, as a model system.
The specific aims of the proposed investigations are to: 1) Isolate mutant RNAI substrates having sequence changes that affect the rate of ams/rne-dependent decay of RNAI in vivo. Identify sequence motifs and other structural features that affect the rate and/or site of cleavage in vivo and determine the effects of these changes on RNAseE-mediated cleavage of RNAI in vitro, 2) Isolate and characterize mutations in the ams/rne gene that alter plasmid copy number by increasing the rate of cleavage of RNAI in vivo; determine the effects of these mutations on other ams/rne-controlled ribonucleolytic activities. Isolate RNAseE from mutated cells and characterize its activity in vitro, 3) Characterize the RNAI cleaving activity from ams/rne ts mutant cells complemented by cloned yeast and mammalian cDNA and elucidate the basis for complementation. Determine the nature of the gene product encoded by the complementing clones, and 4) Isolate E. coli mutants that affect the activity of the growth-rate controlled enzyme that degrades RNAI variants lacking an RNAseE cleavage site; elucidate the basis of the growth rate control. Collectively, these experiments will provide answers to questions that are of fundamental importance to an understanding of the regulatory role of RNA in key biological processes crucial to current efforts to develop approaches that employ antisense and RNA molecules and ribozymes intracellularly in the treatment of disease.
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