STRUCTURE AND GENETIC CONTROL OF COLICINES

该提案涉及遗传和生化分析 抗生素耐药性复制起始的调节 大肠杆菌中的质粒 R6K。 该多拷贝质粒有 38 KB 大小并指定对抗生素氨苄西林的耐药性和 链霉素。 先前的研究已经定义了连续复制 大小跨度为 4 KB 的区域。 在这个地区的三个起源 复制，指定为 Alpha、Beta 和 Gamma，已被识别并 显示在体内和体外复制系统中发挥作用。 在 另外，基因（pir）指定复制起始蛋白（Pi） 位于 Gamma 和 Beta 原点之间，是以下活动所必需的 皆三源。 Pi 蛋白是多功能的，因为它表现出 R6K 复制起始中的正向和负向活性 自动调节 pir 基因的表达。 三人的活动 复制起点还需要顺式 7 个 22 碱基对直接重复序列 位于伽马起源区域。 分子遗传学和生化 分析将旨在确定 Pi 蛋白在 质粒 R6K 起始的调控，重点是 R6K 的性质 Pi 蛋白与直接重复区域、其他片段的相互作用 R6K 复制子和大肠杆菌复制蛋白。 介绍 Pi 蛋白的野生型和突变型以及同向重复序列将是 使用凝胶电泳、电子显微镜和核磁共振进行分析 光谱技术。 突变变化的直接影响 重复区域和Pi蛋白对RNA转录本形成的影响 R6K 复制子和复制也将在体外进行检查。 R6K基因 指导从 Beta 起源开始复制的产品 已被识别并将被表征。 另外分子遗传学 方法将针对pir基因的自动调节机制 表达以及这种自动调节在质粒拷贝数中的作用 控制。 最后，大肠杆菌宿主蛋白在R6K复制中的作用 将通过分离和表征改变的大肠杆菌突变体来探索 野生型和突变型 R6K 质粒的复制特性。 这些 实验方法旨在阐明的主要组成部分 监管机制及其相互作用的性质负责 用于控制质粒 R6K 的拷贝数，该质粒是主要质粒的成员 一组质粒，其特征是在某个位置存在同向重复序列 复制起点和质粒编码的复制蛋白。

This proposal is concerned with a genetic and biochemical analysis of the regulation of initiation of replication of the antibiotic resistance plasmid R6K in Escherichia coli. This multi-copy plasmid is 38 kilobases in size and specifies resistance to the antibiotics ampicillin and streptomycin. Previous studies have defined a contiguous replication region that spans 4 kilobases in size. Within this region three origins of replication, designated Alpha, Beta and Gamma, have been identified and shown to function in vivo and in an in vitro replication system. In addition, a gene (pir) specifies a replication initiation protein (Pi) is located between the Gamma and Beta origins and is required for activity of all three origins. The Pi protein is multi-functional in that it exhibits positive and negative activity in the initiation of R6K replication and autoregulates the expression of the pir gene. The activity of the three replication origins also requires in cis seven 22 base pair direct repeats located in the Gamma-origin region. Molecular genetic and biochemical analysis will be directed at determining the role of the Pi protein in the regulation of initiation of plasmid R6K with emphasis on the nature of the interaction of the Pi protein with the direct repeat region, other segments of the R6K replicon, and E. coli replication proteins. The introduction of wild-type and mutant forms of the Pi protein and the direct repeats will be analyzed using gel electrophoresis, electron microscopy and NMR spectroscopy techniques. The effect of mutational changes in the direct repeat region and the Pi protein on the formation of RNA transcripts of the R6K replicons and replication also will be examined in vitro. An R6K gene product that directs the initiation of replication from the Beta-origin has been identified and will be characterized. In addition molecular genetic approaches will be directed at the mechanism of autoregulation of pir gene expression and the role of this autoregulation in plasmid copy number control. Finally, the role of E. coli host proteins in R6K replication will be explored by isolating and characterizing E. coli mutants that alter the replication properties of wild-type and mutant R6K plasmids. These experimental approaches are designed to elucidate the major components of the regulatory machinery and the nature of their interactions responsible for the control of the copy number of plasmid R6K, a member of a major group of plasmids characterized by the presence of direct repeats at a replication origin and a plasmid encoded replication protein.