STRUCTURE AND GENETIC CONTROL OF COLICINES

Colicines 的结构和遗传控制

• 批准号: 6169541
• 负责人: DONALD R HELINSKI
• 金额: $ 33.69万
• 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/6169541
• 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.

这项提案中的研究涉及到基因的特征 以及负责启动复制的生化机制 以及广泛寄主范围的RK2质粒在 细菌。除了它在确定耐药性方面的医学重要性之外 对常用的抗生素四环素、卡那霉素和氨苄西林 广泛的细菌，质粒RK2作为模型系统 了解DNA复制的启动和分布， 可能是通过一种分割机制，将DNA元件分割到子细胞 在细胞分裂时。RK2编码一种复制起始蛋白(TrfA) 以及具有作为其主要特征的8 17的复制起始序列 碱基对直接重复(迭代)。此外，一个3.2千碱基的片段 该质粒含有两个操纵子，命名为parCBA和Parde，它们稳定地 在大肠埃希氏菌和其他革兰氏阴性菌中保持该质粒。 Parde操纵子编码了一种后隔离杀戮机制， 更正了在不断增长的细菌种群中丢失的质粒，而 ParCBA操纵子通过一种未知的基因提供有效的质粒稳定 可能涉及到质粒分割的机制。建议进行的研究 RK2复制的启动是为了理解 RK2复制起始处的生化事件，包括 TrfA蛋白和寄主DNAA蛋白在沙门氏菌中的作用 开放复合体的形成和TrfA可能的相互作用 与DNABC蛋白复合体和其他宿主复制蛋白结合的蛋白质 来自大肠杆菌和其他细菌。这些研究将同时使用野生型和 TrfA蛋白的突变形式及多种体外技术 包括利用从大肠杆菌中纯化的蛋白质的复制系统。 此外，RK2来源之间的偶联复合体的作用 复制和TrfA蛋白将从以下两个方面进一步评估 目的：探讨质粒拷贝数调控中的结构和作用。分析 RK2稳定区的研究将侧重于阐明其作用机制 由parCBA操纵子和 PARC、B和A蛋白在这一过程中的生化作用。这些 研究可能有助于我们理解基因和 寄主范围广泛复制和稳定维持的生化基础 医学上重要的细菌质粒的性质。