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: 3857015
• 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/3857015
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA replication; DNA replication origin; Escherichia coli; bacterial DNA; bacterial proteins; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic techniques; genetic transcription; microorganism growth; site directed mutagenesis

The long term goal of our research is to understand the mechanisms regulating the initiation of DNA replication. In both prokaryotic and eukaryotic cells, the rate of chromosomal duplication is controlled by the rate of initiation at the origin of replication, not by the rate of chain elongation. Consequently, the determinants of the replication rate and its control are necessarily involved in the initiation step. In the bacterial cell, initiation of replication at the bacterial origin, oriC, requires an RNA polymerase-mediated transcription event and the presence of the DnaA protein. This project seeks to define these events in molecular terms. We are using genetic and biochemical approaches to elucidate both the involvement of RNA polymerase in initiation, whether it be priming or transcriptional activation, and the interaction of DnaA protein with oriC in forming the initial complex. Only with a detailed knowledge of these two events can regulation of DNA replication be understood. In addition, we plan to study the regulation of expression of the dnaA gene, because expression of this gene may be critical in linking the initiation event to the cell cycle. Specifically, we plan to: . Characterize the involvement of sequences adjacent to oriC in initiation of DNA replication. Deletions of regions next to oriC will be introduced into the chromosome and characterized. These deletions will be replaced with transcription termination sequences and G+C blocks. . Establish if the original Fraction II in vitro system requires RNA polymerase. . Define the structural features and other parameters that contribute to forming the oriC initial complex. Does DnaA protein induce bending at one DnaA binding site and what contributions to bending occur when more binding sites are added, in parallel positions or in alternating positions, as is seen in oriC? . Investigate regulation of DnaA protein expression. Use our single round in vitro transcription assay and site-directed mutagenesis to assess sequence specifications for growth rate regulation and other controlling molecules. Search for other factors that regulate dnaA gene expression with gel shift assays and Southwesterns.

我们研究的长期目标是了解 调节DNA复制的起始。 在原核生物和 在真核细胞中，染色体复制的速率由 复制起点的起始速率，而不是链的速率 伸长率 因此，复制率及其 控制必须包括在启动步骤中。 在细菌 细胞，在细菌起源处复制的起始，oriC，需要一个 RNA聚合酶介导的转录事件和DnaA的存在 蛋白 这个项目试图从分子角度来定义这些事件。 我们 正在使用遗传学和生物化学的方法来阐明 RNA聚合酶参与起始，无论是引发还是 转录激活，以及DnaA蛋白与oriC的相互作用 形成最初的复合体。 只有对这些有详细的了解 有两个事件可以理解DNA复制的调节。 此外，本发明还提供了一种方法， 我们计划研究dnaA基因表达的调控，因为 该基因的表达可能在将起始事件与 细胞周期 具体而言，我们计划： . 描述oriC附近序列参与的特征， 启动DNA复制。 删除oriC旁边的区域将 将其引入染色体并表征。 这些缺失 将替换为转录终止序列和G+C 个街区. .确定原始组分II体外系统是否需要RNA 聚合酶。 .定义结构特征和其他参数， 形成oriC初始复合物。 DnaA蛋白质在一点诱导弯曲吗 DnaA结合位点和什么贡献弯曲发生时，更多的结合 以平行位置或交替位置添加位点，如 在OriC中看到？ .研究DnaA蛋白表达的调控。 用我们的一发子弹 体外转录测定和定点诱变以评估 生长速率调节和其他控制的序列规范 分子。 寻找调节dnaA基因表达的其他因素 凝胶迁移分析和西南部的。