STRUCTURE AND GENETIC CONTROL OF COLICINES

Colicines 的结构和遗传控制

• 批准号: 2429346
• 负责人: DONALD R HELINSKI
• 金额: $ 29.97万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-02-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2429346
• 关键词: DNA binding protein; DNA replication; Escherichia coli; Pseudomonas; R factors; bacterial genetics; bacterial proteins; colicines; drug resistance; gel electrophoresis; gene deletion mutation; gene induction /repression; genetic strain; gram negative bacteria; molecular cloning; nucleic acid sequence; operon; protein purification; protein structure function

The research in this proposal deals with the characterization of genetic and biochemical mechanisms responsible for the initiation of replication and the stable maintenance of the broad host-range plasmid RK2 in bacteria. In addition to its medical importance in specifying resistance to the commonly used antibiotics tetracycline, kanamycin and ampicillin in a wide range of bacteria, plasmid RK2 serves as a model system for understanding the initiation of DNA replication and the distribution, possibly by a partitioning mechanism, of DNA elements to daughter cells upon cell division. RK2 encodes a replication initiation protein (TrfA) and a replication origin sequence that has as its main feature eight 17 base pair direct repeats (iterons). In addition, a 3.2 kilobase segment of the plasmid contains two operons, designated parCBA and parDE, that stably maintain the plasmid in Escherichia coli and other Gram-negative bacteria. The parDE operon encodes a post-segregational killing mechanism that corrects for loss of the plasmid in a growing population of bacteria while the parCBA operon provides effective plasmid stabilization by an unknown mechanism that may involve plasmid partitioning. The proposed studies of the initiation of RK2 replication are directed at understanding biochemical events at the RK2 origin of replication including the contribution of the TrfA protein and the host DnaA protein to the formation of an open complex and the possible interaction of the TrfA protein with the DnaBC protein complex and other host replication proteins from E. coli and other bacteria. These studies will use both wild-type and mutant forms of the TrfA protein and a variety of in vitro techniques including a replication system utilizing purified proteins from E. coli. Furthermore, the role of coupled complexes between RK2 origins of replication and the TrfA protein will be further assessed both with regard to structure and role in regulation of plasmid copy-number. The analysis of the RK2 stabilization region will emphasize elucidating the mechanism of plasmid stable maintenance encoded by the parCBA operon and the biochemical roles of the ParC, B and A proteins in this process. These studies are likely to contribute to our understanding of the genetic and biochemical basis of broad host-range replication and stable maintenance properties of medically important bacterial plasmids.

本提案中的研究涉及遗传特征 和负责复制启动的生化机制 以及广泛宿主范围质粒 RK2 的稳定维持 细菌。除了其在确定耐药性方面的医学重要性 常用抗生素有四环素、卡那霉素、氨苄青霉素 质粒 RK2 作为多种细菌的模型系统 了解 DNA 复制的起始和分布， 可能通过DNA元件到子细胞的分配机制 细胞分裂时。 RK2 编码复制起始蛋白 (TrfA) 以及以八个为主要特征的复制起点序列 17 碱基对直接重复序列（迭代子）。此外，还有一个 3.2 kb 的段 该质粒包含两个操纵子，分别命名为 parCBA 和 parDE，它们可以稳定地 将质粒保留在大肠杆菌和其他革兰氏阴性细菌中。 parDE 操纵子编码一种分离后杀伤机制， 纠正不断增长的细菌群体中质粒的丢失，同时 parCBA 操纵子通过未知的作用提供有效的质粒稳定性 可能涉及质粒分割的机制。拟议的研究 RK2复制的启动旨在理解 RK2 复制起点的生化事件包括 TrfA 蛋白和宿主 DnaA 蛋白对 开放复合物的形成以及 TrfA 可能的相互作用 具有 DnaBC 蛋白复合物和其他宿主复制蛋白的蛋白 来自大肠杆菌和其他细菌。这些研究将使用野生型和 TrfA 蛋白的突变形式和各种体外技术 包括利用大肠杆菌纯化蛋白的复制系统。 此外，RK2 起源之间的偶联复合物的作用 复制和 TrfA 蛋白将进一步评估 结构和在质粒拷贝数调节中的作用。分析 RK2稳定区域的研究将重点阐明其机制 parCBA 操纵子编码的质粒稳定维持和 ParC、B 和 A 蛋白在此过程中的生化作用。这些 研究可能有助于我们了解遗传和 广泛宿主范围复制和稳定维持的生化基础 医学上重要的细菌质粒的特性。