STRUCTURE AND GENETIC CONTROL OF COLICINES

这项提案中的研究涉及遗传学的表征， 以及负责复制起始的生化机制 以及广宿主范围质粒RK2在 细菌除了它在医学上的重要性， 常用的抗生素四环素，卡那霉素和氨苄青霉素， 广泛的细菌，质粒RK2作为模型系统， 了解DNA复制的起始和分布， 可能是通过一种分配机制，将DNA元件分配给子细胞 在细胞分裂时。RK2编码复制起始蛋白（TrfA） 和一个复制起始序列，其主要特征是8个17 碱基对直接重复序列（iteron）。此外，3.2内切酶片段 该质粒含有两个操纵子，称为parCBA和parDE，它们稳定地 在大肠杆菌和其它革兰氏阴性菌中维持质粒。 parDE操纵子编码分离后杀伤机制， 在不断增长的细菌种群中校正质粒的丢失， parCBA操纵子通过一种未知的 可能涉及质粒分配的机制。拟议的研究 RK2复制起始是为了理解 RK2复制起点的生化事件，包括 TrfA蛋白和宿主DnaA蛋白对细胞凋亡的贡献 开放复合物的形成和TrfA的可能相互作用 蛋白质与DnaBC蛋白质复合物和其它宿主复制蛋白质 来自大肠大肠杆菌和其他细菌。这些研究将使用野生型和 TrfA蛋白的突变形式和多种体外技术 包括利用来自E.杆菌 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， 质粒拷贝数调控的结构和作用。分析 RK2稳定区的研究将强调阐明 由parCBA操纵子编码的质粒稳定维持和 ParC、B和A蛋白在该过程中的生物化学作用。这些 这些研究可能有助于我们理解遗传学， 广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性质。

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.