INITIATION OF DNA REPLICATION--DNAA, RNA POLYMERASE, & COUPLING TO CELL CYCLE

DNA复制的起始--DNAA、RNA聚合酶、

• 批准号: 3756464
• 负责人: JUDITH W ZYSKIND
• 金额: --
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3756464
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA replication; DNA replication origin; Escherichia coli; bacterial DNA; bacterial proteins; cell cycle; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic techniques; genetic transcription; microorganism growth; protein structure function; receptor coupling; site directed mutagenesis

The long term goal of this project is to understand how initiation of DNA replication is coupled to growth rate. Our objective is to understand how DNA replication, cell growth, and cell division are so precisely and coordinately regulated. In both prokaryotic and eukaryotic cells, the rate of chromosomal duplication is controlled by the rate of initiation at the origin(s) of replication, not by the rate of chain elongation. The rate-limiting steps of the initiation event remain unidentified. To understand how the frequency of the initiation event is determined, the mechanisms that regulate the expression of the DnaA protein must be understood. Both genetics and biochemistry are used to elucidate the regulatory circuits controlling expression of the dnaA operon promoters. The major dnaA promoter is growth rate regulated and under stringent control. Guanosine 3',5'-bispyrophosphate(ppGpp) may provide the molecular basis for growth rate regulation of DnaA protein expression. Mutations in this promoter and in the dnaA box nearby that eliminate promoter activity and binding of DnaA protein to the dnaA box will be introduced into the chromosome and assayed for growth rate regulation and stringent control in order to identify which regulatory sequences affect growth rate regulation and stringent control. A single round in vitro transcription assay is being used to ascertain the contribution of ppGpp and DnaA protein to regulation of the activity of each of the dnaA promoters. Mutants defective in the inhibitory response to ppGpp will be examined for growth rate regulation and precise timing of initiation of DNA replication in the cell cycle. We will determine whether the dnaA gene is still under growth rate control in cells that lack ppGpp -- is there a growth rate regulation mechanism other than ppGpp that controls the expression of DnaA protein? As in the past, MBRS students will be involved in both the technical and scientific aspects of the research.

这个项目的长期目标是了解DNA的启动是如何 复制与增长率相关联。 我们的目标是了解 DNA复制、细胞生长和细胞分裂是如何精确地 协调调控。 在原核和真核细胞中， 染色体复制的速率受起始速率的控制 在复制的起点，而不是由链延伸的速率。 起始事件的限速步骤仍未确定。 到 了解启动事件的频率是如何确定的， 调节DnaA蛋白表达的机制必须是 明白 遗传学和生物化学都被用来阐明 控制dnaA操纵子启动子表达的调节回路。 主要的dnaA启动子是生长速率调节的，并且在严格的 控制 鸟苷3 '，5'-双焦磷酸（ppGpp）可以提供 DnaA蛋白表达的生长速率调节的分子基础。 在这个启动子和附近的dnaA盒中的突变， 启动子活性和DnaA蛋白与dnaA盒的结合将是 引入染色体并测定生长速率调节， 为了鉴定哪些调节序列影响 增长速度调控和严格控制。体外单轮 转录测定被用来确定ppGpp的贡献 和DnaA蛋白质的活性调节， 发起人。 在对ppGpp的抑制反应中有缺陷的突变体将 检查生长速率调节和启动的精确时间 在细胞周期中的DNA复制。 我们将确定DNA是否 在缺乏ppGpp的细胞中， 除了ppGpp之外，还有一种增长率调节机制， DnaA蛋白的表达 与过去一样，MBRS学生将参与技术和 研究的科学方面。