Genetic and Biochemical Analysis of a Peptide Export System

提出的实验目的是阐明细菌中肽和蛋白质输出的机制。具体地说，要使用的系统是出口微菌素B17(MccB17)，这是一种由大肠杆菌生产的多肽抗生素。六个基因，从mcbB到mcbG，是产生和免疫MccB17所必需的，MccB17本身就是MCBA的产物。这种抗生素作用于敏感的肠道细菌细胞，以阻止DNA复制。McbE和mcbF基因产物似乎介导了MccB17的输出；mcbE和mcbF的突变及其基因外抑制物将描述MccB17输出机制中这些组件的作用、相互作用和功能域。细胞内和细胞外MccB17将通过生化和细胞分级研究以及电子显微镜来表征。MccB17分子中阻止其出口的突变将识别运输的功能区域；这些允许改变的多肽输出的基因外抑制物将鉴定输出的多肽与其输出所需的蛋白质之间的相互作用。%通过妇女研究机会研究启动奖，斯克维斯基博士确立了自己作为独立研究员的地位。这项工作可能会很有趣，因为在大肠杆菌中，将蛋白质输出到细胞外部的情况很少见。在这些研究中获得的信息将对细菌克隆基因产品的商业生产具有许多可能的实际应用。

The objective of the experiments proposed is to elucidate mechanisms of peptide and protein export in bacteria. Specifically, the system to be used is the export of Microcin B17 (MccB17), a peptide antibiotic produced by E. coli. Six genes, mcbB through mcbG, are necessary for production of and immunity to MccB17, itself the product of mcbA. The antibiotic acts on sensitive enteric bacterial cells to block DNA replication. The mcbE and mcbF gene products appear to mediate export of MccB17; mutations in mcbE and mcbF and extragenic suppressors thereof will delineate the roles, interactions, and functional domains of these components of the MccB17 export machinery. Intracellular and extracellular MccB17 will be characterized by biochemical and cell fractionation studies and by electron microscopy. Mutations in the MccB17 molecule that prevent its export will identify functional domains for transport; extragenic suppressors of these mutations that allow export of the altered peptide will identify interactions between the exported peptide and the proteins necessary for its export.%%% Dr. Skvirsky is establishing herself as an independent investigator through this Research Opportunities for Women Research Initiation award. The work promises to be interesting because export of proteins to the exterior of the cell is rare in the bacterium E.coli. The information obtained in these studies will have numerous possible practical applications to the commercial production of products of cloned genes in bacteria.***//