In Escherichia coli messenger RNAs are rapidly degraded with in average functional half-life of approximately 90 seconds. Based on our recent results and those from other laboratories, we have developed a working model for mRNA turnover in which functional mRNAs are degraded in a series of apparent endonucleolytic steps. The initial breakdown products are subsequently digested exonucleolytically to mononucleotides which can be recycled for the synthesis of new RNA species. By employing a multiple ams pnp rnb mutant we have been able to stabilize the initial breakdown products of a number of individual mRNA molecules. Our long range goal is to understand the important regulatory features of mRNA turnover along with its enzymology. In the previous grant period we succeeded in showing that ribonuclease II, polynucleotide phosphorylase, and the Ams gene product are essential for the later steps of mRNA turnover. In addition, we were able to identify a new locus (mrd, messenger RNA degradation), that is required for both mRNA degradation and cell viability. Our current aims are to analyze the fate of individual mRNA molecules in strains deficient in polynucleotide phosphorylase, ribonuclease II and the ams gene product. By sequencing the ends of the initial breakdown products we hope into identify the structural features associated with the cleavage sites. Additionally, we will attempt to clone and identify by the Ams and Mrd gene products. New multiple mutants carrying mutations in the ams mrd pnp rnb and rnc genes will be constructed and their mRNA turnover patterns determined. We will also purify and characterize both temperature sensitive ribonuclease II and ribonuclease III proteins. If time permits, we will attempt to gain further insights into the mechanism of mRNA turnover by examining temperature resistant revertants of ams pnp rnb and pnp rnb multiple mutants.
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