How Does RNase III Regulate Antibiotic Production in Streptomyces Coelicolor?

临床和兽药中使用的所有抗生素中，近70%是由链霉菌属细菌合成的。核糖核酸酶III （RNase III）是广泛存在于细菌和真核生物中的一种双链特异性核糖核酸内酶。在链霉菌的RNase III基因突变减少或取消所有四种抗生素的合成通常由生物体产生。因此，RNase III是葡萄球菌合成抗生素的全球性调节因子。RNA免疫沉淀和微阵列分析（一种RIP-Chip方法）将用于研究RNase III调控抗生素产生的机制。针对RNase III突变形式的抗体将用于沉淀酶与其靶rna之间形成的复合物。rna将从免疫沉淀中提取并用于合成互补dna （cdna）。这些cdna将用于微阵列研究，以鉴定在免疫沉淀中富集的rna。对可能参与基因表达调控的RNA，如RNA聚合酶sigma因子、抑制因子、激活因子等的mrna将进一步研究。裂解实验将证实这些mrna是RNase III的底物。相应的基因将被破坏，并评估破坏对抗生素生产的影响。通过这种方式，将鉴定出其产物为RNase III底物并调节葡萄球菌抗生素生产的基因。许多计划中的实验将适合本科生和本科生研究人员，特别是少数民族和女性本科生，将被招募到该项目中。该研究还将涉及申请人实验室与英国实验室之间的合作，进一步促进国家科学基金会促进国际研究合作的目标。该项目还将为相关博士后提供良好的培训机会。研究结果将传播到科学界，扩大我们对生产有用抗生素的机制的理解。

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.