How Does RNase III Regulate Antibiotic Production in Streptomyces Coelicolor?

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Nearly 70% of all antibiotics used in clinical and veterinary medicine are synthesized by members of the bacterial genus, Streptomyces. Ribonuclease III (RNase III) is a double strand specific endoribonuclease distributed widely in bacteria and eukaryotes. Mutations in the RNase III gene in Streptomyces coelicolor reduce or abolish synthesis of all four antibiotics normally produced by the organism. Thus, RNase III is a global regulator of antibiotic synthesis in S. coelicolor. RNA immunoprecipitation followed by microarray analysis (a RIP-Chip approach) will be used to examine the mechanism of RNase III regulation of antibiotic production. Antibody to a mutant form of RNase III will be used to precipitate complexes formed between the enzyme and its target RNAs. RNAs will be extracted from the immunoprecipitates and used to synthesize complementary DNAs (cDNAs). The cDNAs will be used in microarray studies to identify RNAs that are enriched in the immunoprecipitates. RNAs that may be involved in the regulation of gene expression, e.g. mRNAs for RNA polymerase sigma factors, repressors, activators, etc. will be studied further. Cleavage assays will verify that these mRNAs are substrates for RNase III. The corresponding genes will be disrupted and the effects of disruption on antibiotic production assessed. In this way, genes whose products are substrates for RNase III and which regulate antibiotic production in S. coelicolor will be identified.Many of the planned experiments will lend themselves to performance by undergraduates and undergraduate researchers, especially minority and women undergraduates, will be recruited to the project. The research will also involve collaboration between the applicant lab and a lab in the UK, furthering the NSF goal of fostering international research collaborations. The project will also provide an excellent training opportunity for the postdoctoral fellows associated with it. Results of the research will be disseminated to the scientific community extending our understanding of the mechanisms involved in the production of useful antibiotics.