STRUCTURE AND GENETIC CONTROL OF COLICINES

Colicines 的结构和遗传控制

• 批准号: 3124345
• 负责人: DONALD R HELINSKI
• 金额: $ 31.28万
• 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 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124345
• 关键词: DNA replication; DNA replication origin; Escherichia coli; Pseudomonas aeruginosa; R factors; Rhizobiaceae; ampicillin; bacterial DNA; bacterial genetics; binding proteins; cell cycle; cell free system; colicines; gene expression; gene induction /repression; genetic manipulation; genetic promoter element; genetic strain; gram negative bacteria; kanamycin; laboratory rabbit; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; nucleic acid sequence; open reading frames; protein structure function; radiotracer; streptomycin

The research in this proposal deals with the characterization of genetic and biochemical mechanisms responsible for the regulation of replication and for stable maintenance of the broad-host-range plasmid RK2 in bacteria. This plasmid specifies resistance to the antibiotics tetracycline, ampicillin and kanamycin and is stably maintained in the extrachromosomal state in a wide range of Gram-negative bacteria. Two components of this plasmid that are absolutely required for its replication are the trfA gene that encodes two replication initiation proteins (44 kDa and 33 kDa), the smaller of which is the result of an internal translational start in the same open reading frame, and a replication origin sequence that contains as its main feature eight 17 bp repeats (iterons) in groups of five and three. The 44 kDa and 33 kDa proteins are essentially equivalent in activity in the bacterium Escherichia coli and both proteins specifically bind to the iterons at the RK2 replication origin. The major thrust of this proposal is to examine the nature of the interactions between the TrfA replication protein(s) and the sequence at the replication origin that are responsible for the regulation of initiation of replication and the ability of this antibiotic resistance plasmid to be maintained in a wide range of bacteria. These studies also will be concerned with analyses of the interactions between the TrfA protein/origin sequence complex and specific host replication proteins isolated from several Gram-negative bacteria. The analyses will be carried out in vivo and in vitro and will involve both wild-type and mutant TrfA proteins, including defective and copy-up mutants. An attempt will be made to identify regions of the TrfA protein responsible for its various interactions with the replication origin and the host replication proteins. Finally, a region of the RK2 plasmid that appears to encode a broad-host-range plasmid partitioning function will be examined in order to understand the mechanism(s) responsible for the partitioning of a plasmid to daughter cells upon cell division. In addition to providing information on the regulation of initiation of replication and the stable maintenance of an extrachromosomal element in bacteria, a practical fallout of this basic study will be the construction of high-copy number and stably maintained plasmid vectors for gene cloning in a wide range of Gram-negative bacteria of medical and agricultural importance.

本提案中的研究涉及遗传特征 和负责复制调节的生化机制 并用于稳定维持广泛宿主范围的质粒 RK2 细菌。 该质粒指定对抗生素的抗性 四环素、氨苄西林和卡那霉素，并稳定地维持在 多种革兰氏阴性细菌中的染色体外状态。 二 该质粒的组成部分是其绝对必需的 复制是编码两个复制起始的 trfA 基因 蛋白质（44 kDa 和 33 kDa），其中较小的一个是 内部翻译起始于同一开放阅读框，并且 复制起点序列，其主要特征为 8 个 17 bp 以五组和三组重复（迭代）。 44 kDa 和 33 kDa 蛋白质在细菌中的活性基本相同 大肠杆菌和两种蛋白质在以下位置特异性结合到迭代子上： RK2复制起点。 该提案的主要目的是 检查 TrfA 复制之间相互作用的性质 蛋白质和复制起点的序列是 负责复制起始的调节和 该抗生素抗性质粒能够在广泛的环境中维持 细菌范围。 这些研究还将涉及以下方面的分析： TrfA 蛋白/起始序列复合物之间的相互作用 从几种革兰氏阴性菌中分离出的特定宿主复制蛋白 细菌。 分析将在体内和体外进行，并将 涉及野生型和突变型 TrfA 蛋白，包括有缺陷的和 复制突变体。将尝试确定 TrfA 的区域 负责其与复制的各种相互作用的蛋白质 起源和宿主复制蛋白。 最后是RK2的一个区域 似乎编码广泛宿主范围的质粒分配的质粒 将检查功能以了解其机制 负责将质粒分配给子细胞 分配。 除了提供有关监管的信息外 复制的启动和稳定的维持 细菌中的染色体外元素，这一基本的实际后果 研究将构建高拷贝数并稳定维护 用于多种革兰氏阴性菌基因克隆的质粒载体 具有医学和农业重要性的细菌。