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.

该项目的长期目标是了解 DNA 的启动过程 复制与增长率相关。 我们的目标是了解 DNA复制、细胞生长和细胞分裂如何如此精确和 统筹监管。 在原核细胞和真核细胞中， 染色体复制率由起始率控制 在复制起点，而不是链伸长率。 启动事件的速率限制步骤仍未确定。 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 明白了。 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电路。 主要的 dnaA 启动子的生长速率受严格控制 控制。 鸟苷 3',5'-双焦磷酸 (ppGpp) 可以提供 DnaA 蛋白表达生长速率调节的分子基础。 该启动子和附近 dnaA 盒中的突变消除了 启动子活性以及 DnaA 蛋白与 dnaA 盒的结合将 引入染色体并进行生长速率调节分析 严格控制以确定哪些调控序列影响 增速调控，严格控制。体外单轮 转录测定用于确定 ppGpp 的贡献 和 DnaA 蛋白来调节每个 dnaA 的活性 发起人。 对 ppGpp 的抑制反应有缺陷的突变体将 检查生长速率调节和精确的启动时间 细胞周期中 DNA 复制的过程。 我们将确定 dnaA 是否 在缺乏 ppGpp 的细胞中，该基因仍处于生长速率控制之下—— 除了 ppGpp 之外，还有其他增长率调节机制可以控制 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。