GENETIC FUNCTIONS OF CONJUGATIVE STREPTOCOCCAL R FACTOR

描述：这项建议涉及一项分子和基因分析 调控接合蛋白表达的调控机制 四环素耐药质粒pCF10在肠球菌中的转移 粪便。对这一系统的分析表明，质粒编码的 供体细胞中的传递函数是由非常少量的 受体细胞产生的多肽信息素。当前可用的数据具有 导致制定了两个监管重要阶段的模型 这个系统。描述信息素区隔的模型 内化机制解释了供体细胞装载的能力 交配对受体产生的信息素的反应，并避免 内源信息素的自我诱导。一种参与的模式 信息素在胞内阶段，这涉及到积极控制 此外，还介绍了翻译方法。拟议中的实验旨在 测试模型的重要预测，包括以下目标： I)确定推测的产生内源性信息素的前信息素分子 活性；ii)使用遗传学、免疫电子显微镜和 生物化学研究细胞内不同成分的位置 内化机制及其相互作用；iii)使用组合遗传 和生化方法分析一种质粒编码的相互作用 调控的RNA分子及其预测的核糖体靶标，以及其他 系统的组件和iv)使用生化技术来阐明 一种关键的负性调节蛋白PRGX的作用机制 系统。

DESCRIPTION: This proposal involves a molecular and genetic analysis of the regulatory mechanisms involved in controlling expression of conjugative transfer of the tetracycline-resistance plasmid, pCF10 in Enterococcus faecalis. Analysis of this system has revealed that plasmid-encoded transfer functions in donor cells are induced by very small amounts of a peptide pheromone produced by recipient cells. Currently available data has led to the formulation of two models for important stages of regulation in the system. A model describing a compartmentalization of pheromone internalization machinery accounts for the ability of a donor cell to mount mating response to pheromone produced by recipients, and avoid self-induction by endogenous pheromone. A model for the involvement of pheromone at an intracellular stage, which involves positive control of translation is also presented. The experiments proposed are designed to test important predictions of the models, and include the following aims: i) to identify the putative propheromone molecule that generates endogenous activity; ii) use a combination of genetics, immunoelectron microscopy, and biochemistry to study the cellular location of various components of the internalization machine and their interactions; iii) use a combined genetic and biochemical approach to analyze the interactions of a plasmid-encoded regulatory RNA molecule with its predicted ribosomal target, and other components of the system and iv) use biochemical techniques to elucidate the mechanisms of action of a key negative regulatory protein, PrgX, in the system.