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.

该提案中的研究涉及遗传的表征 以及负责复制调节的生化机制 并稳定维护宽主范围质粒RK2 细菌。 该质粒指定对抗生素的抗性 四环素，氨苄青霉素和卡纳米霉素，并稳定地保持在 在革兰氏阴性细菌范围内，外染色体态。 二 该质粒的成分绝对需要 复制是编码两个复制启动的TRFA基因 蛋白质（44 kDa和33 kDa），其中较小是由 内部翻译在相同的开放阅读框架中开始 复制起源序列，其中包含其主要特征八17 bp 重复（Iterons），分别为五个和三组。 44 kDa和33 kDa 蛋白质在细菌的活性上基本上是等效的 大肠杆菌和两种蛋白质特异性结合在 RK2复制起源。 该提议的主要目的是 检查TRFA复制之间相互作用的性质 蛋白质和复制起源处的序列 负责调节复制的开始和 这种抗生素抗性质粒的能力保持在宽度 细菌范围。 这些研究还将与 TRFA蛋白/起源序列复合物与 从几个革兰氏阴性分离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，包括缺陷和 复制突变体。将尝试确定TRFA的区域 蛋白质负责其与复制的各种相互作用 原点和宿主复制蛋白。 最后，RK2区域 质粒似乎编码了宽大的质范围质粒分配 将检查功能以了解机制 负责将质粒分配到细胞上的子细胞 分配。 除了提供有关法规的信息 复制的启动和稳定的维护 细菌中的外染色体元素，该基本的实际影响 研究将是构造高拷贝数，并稳定维护 基因克隆的质粒载体在跨革兰氏阴性含量中 医学和农业重要性的细菌。