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图库曼大学与劳尔·所罗门合作进行，作为NIH#R01GM64530号补助金的延伸。长期目标是了解21个氨基酸的抗生素Microcin J25(MccJ25)的结构、功能和成熟度。我们以前的工作表明，MccJ25是由58个氨基酸长的McjA前体在McjB和McjC蛋白的作用下产生的，它针对细菌RNA聚合酶。进一步的研究表明，Mccj25有一个独特的结构折叠--螺纹套索。美国和阿根廷集团的互补优势将结合在一起，以实现以下具体目标： 1.建立MccJ25分子的结构-功能关系。将对质粒生的mcjA基因的MccJ25部分进行系统的定点和随机突变。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，细胞在培养基中击败突变的mcjA质粒产生突变的MccJ25的能力将被建立；MccJ25的突变体将被纯化，其抗菌和转录抑制功能将分别通过体内和体外实验来确定。还将寻找对野生型MccJ25耐药细胞具有活性的MccJ25突变体。 2.建立MccJ25体外成熟体系。将制备纯化的重组McjA前体和McjB和McjC，并用于寻找将McjA高效成熟为MccJ25的条件。这项工作的结果将阐明MccJ25的合成、结构功能以及与其靶标的相互作用的细节，从而为转录机制提供新的见解。对MccJ25成熟度的了解可能允许使用基于McjA的嵌合蛋白来构建具有潜在生物学和药理学特性的新型环肽和/或蛋白质。

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.