Structure-function of RNAP inhibitor microcin J25

描述（由申请人提供） 这项研究将主要在阿根廷的图库曼大学与Raul Salomon合作进行，作为NIH资助#R01GM64530的延伸。长期目标是了解21个氨基酸抗生素Microcin J25（MccJ25）的结构，功能和成熟。我们以前的工作表明，MccJ25，这是从一个58个氨基酸长的McjA前体产生的McjB和McjC蛋白的作用靶向细菌RNA聚合酶。进一步的研究表明，Mccj25有一个独特的结构折叠--一个螺纹套索。美国和阿根廷集团的互补优势将结合起来，以实现以下具体目标： 1.建立MccJ25分子的结构-功能关系。将对质粒产生的mcjA基因的MccJ25部分进行系统性位点特异性和随机诱变。在含有mcjBC成熟基因和mcjD免疫基因的互补质粒存在的情况下，将确定细胞在培养基中击败突变mcjA质粒产生突变MccJ25的能力;将纯化MccJ25突变体，并分别通过体内和体外试验确定其抗菌和转录抑制功能。还将寻找对野生型MccJ25具有抗性的细胞具有活性的MccJ25突变体。 2.建立MccJ25体外成熟培养体系。将制备纯化的重组McjA前体以及McjB和McjC，并用于寻找McjA有效成熟为MccJ25的条件。 这项工作的结果将阐明MccJ25合成，结构-功能及其与靶点相互作用的细节，从而对转录机制有新的认识。MccJ25成熟的理解可以允许使用McjA为基础的嵌合蛋白质，构建新的环肽和/或蛋白质具有潜在的有趣的生物学和药理学特性。

DESCRIPTION (provided by applicant) This research will be done primarily in University of Tucuman, Argentina in collaboration with Raul Salomon as an extension of NIH grant # R01GM64530. The long-term goal is to understand the structure, function and maturation of 21 amino acid antibiotic Microcin J25 (MccJ25). Our previous work showed that MccJ25, which is produced from a 58 amino acid-long McjA precursor by the action of McjB and McjC proteins targets bacterial RNA polymerase. Further research showed that Mccj25 has a unique structural fold--a threaded lasso. The complementary strengths of the US and Argentinian groups will be combined to address the following specific aims: 1. To establish structure-functional relationships in MccJ25 molecule. Systematic site-specific and random mutagenesis of the MccJ25 moiety of the plasmid-born mcjA gene will be performed. The ability of cells beating mutant mcjA plasmids to produce mutant MccJ25 in the cultured medium in the presence of a complementary plasmid bearing mcjBC maturation genes and mcjD immunity gene will be established; MccJ25 mutants will be purified and their antibacterial and transcription inhibitory functions will be established by in vivo and in vitro tests, respectively. MccJ25 mutants active against cells resistant to wild-type MccJ25 will also be sought. 2. To establish an in vitro system for MccJ25 maturation. Purified recombinant McjA precursor and McjB and McjC will be prepared and used to find conditions for efficient maturation of McjA into MccJ25. The results of this work will clarify the details of MccJ25 synthesis, structure-function and interaction with its target and thus will allow new insights into the mechanism of transcription. Understanding of MccJ25 maturation may allow to use McjA-based chimeric proteins to construct novel cyclic peptides and or proteins with potentially interesting biological and pharmacological properties.