How Does RNase III Regulate Antibiotic Production in Streptomyces Coelicolor?

在临床和兽医学中使用的所有抗生素中，近70%是由细菌属的成员链霉菌合成的。核糖核酸酶III（Ribonuclease III，RNase III）是一种广泛存在于细菌和真核生物中的双链特异性核糖核酸内切酶。天蓝色链霉菌中RNase III基因的突变减少或消除了生物体正常产生的所有四种抗生素的合成。因此，RNase III是S.天蓝色。RNA免疫沉淀，然后进行微阵列分析（RIP-Chip方法）将用于检查RNase III调节抗生素生产的机制。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。将从免疫沉淀物中提取RNA并用于合成互补DNA（cDNA）。cDNA将用于微阵列研究，以鉴定在免疫沉淀物中富集的RNA。将进一步研究可能参与基因表达调控的RNA，例如RNA聚合酶σ因子、阻遏物、激活物等的mRNA。切割试验将验证这些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。以这种方式，其产物是RNase III的底物并调节S.许多计划中的实验将适合本科生和本科生研究人员的表现，特别是少数民族和女本科生，将被招募到该项目。该研究还将涉及申请实验室和英国实验室之间的合作，进一步促进NSF促进国际研究合作的目标。该项目还将为与之相关的博士后研究员提供一个极好的培训机会，研究结果将传播给科学界，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.