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: 3778401
• 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/3778401
• 关键词: DNA binding protein; DNA directed RNA polymerase; DNA replication; DNA replication origin; Escherichia coli; bacterial DNA; bacterial proteins; gel mobility shift assay; 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其机制 调节 DNA 复制的起始。 在原核生物和 真核细胞中，染色体复制率由 复制起点的起始速率，而不是链的速率 伸长。 因此，复制率的决定因素及其 控制必然参与启动步骤。 在细菌中 细胞，在细菌起源 oriC 处开始复制，需要 RNA 聚合酶介导的转录事件和 DnaA 的存在 蛋白质。 该项目试图用分子术语来定义这些事件。 我们 正在使用遗传和生化方法来阐明 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 形成初始复合体。 只有详细了解这些 可以通过两个事件来了解 DNA 复制的调节。 此外， 我们计划研究 dnaA 基因的表达调控，因为 该基因的表达可能对于将起始事件与 细胞周期。 具体来说，我们计划： 。 表征与 oriC 相邻的序列的参与 DNA复制的起始。 删除 oriC 旁边的区域将 被引入染色体并进行表征。 这些删除 将被转录终止序列和G+C取代 块。 。确定原始 Fraction II 体外系统是否需要 RNA 聚合酶。 。定义有助于的结构特征和其他参数 形成oriC初始复合体。 DnaA 蛋白会诱导弯曲吗 DnaA 结合位点以及更多结合时对弯曲的贡献 按原样以平行位置或交替位置添加站点 在oriC中看到过吗？ 。研究 DnaA 蛋白表达的调节。 使用我们的单轮 体外转录测定和定点诱变来评估 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 与凝胶位移测定和西南。