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

这个项目的长期目标是了解DNA是如何启动的 复制与增长速度相结合。我们的目标是了解 DNA复制、细胞生长和细胞分裂是如何如此精确和 协调监管。在原核和真核细胞中， 染色体复制的速度受起始速度的控制 在复制的起点(S)，不受链条拉长的速度影响。 启动事件的限速步骤仍未确定。至 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解释 控制DNAA操纵子启动子表达的调控电路。 DNAA的主要启动子是生长速度受调控的，并且在严格的 控制力。鸟苷3‘，5’-二焦磷酸(PpGpp)可提供 生长速度调控DNAA蛋白表达的分子基础。 这个启动子和附近的DNAA盒中的突变消除了 Dna A蛋白的启动子活性和与Dna A盒的结合 被导入染色体并进行生长速度调节和检测 严格控制，以确定哪些调控序列会影响 调控增长速度，从严管控。一轮体外试验 转录实验正在被用来确定ppGpp的贡献 和DNAA蛋白来调节每个DNAA的活性 推动者。对ppGpp的抑制反应有缺陷的突变株将 检查生长速度调节和启动的准确时间 在细胞周期中的DNA复制。我们将确定DNAA是否 在缺乏ppGpp-IS的细胞中，基因仍处于生长速度控制之下 除了ppGpp之外，还有一个增长速度调节机制来控制 DNAA蛋白的表达？ 与过去一样，MBRS的学生将同时参与技术和 这项研究的科学方面。

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.