TERMINATION OF DNA REPLICATION IN ESCHERICHIA COLI

大肠杆菌中 DNA 复制的终止

• 批准号: 3467911
• 负责人: THOMAS M. HILL
• 金额: $ 10.95万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467911
• 关键词: DNA binding protein; DNA footprinting; DNA replication origin; Escherichia coli; aminoacid analyzer; bacterial genetics; genetic terminator element; mutant; nucleic acid sequence; site directed mutagenesis; transcription factor; transcription termination

The experiments presented in this proposal are designed to provide a better understanding of the biochemical basis of termination of DNa replication in Escherichia coli. Two components of the E. coli termination system have been identified: a DNA component, which consists of a 23 basepair sequence known as the terminator sequence, and a protein component called Tus, which binds to the DNA terminator sequence. It is proposed to investigate the protein-DNA interactions between Tus and the replisome. Specifically, it is proposed to: 1. Purify and characterize the Tus protein using column chromotography techniques. 2. Study the binding interactions between wild-type Tus protein and the chromosomal terminator sequences using a filter-binding assay. 3. Systematically analyze the sequence requirements for binding of Tus to the terminator sequences by generating site-specific mutations and determining the effect these mutations have on binding. A correlation between binding affinity and termination activity will also be established. 4. Generate mutant Tus proteins to define the active sites for DNA binding and termination. 5. Study the mechanism of replication inhibition using genetic and biochemical methods.

本提案中提出的实验旨在提供一个 更好地理解终止DNa的生化基础 在大肠杆菌中复制。 E.杆菌 终止系统已被确定：一个DNA成分，它包括 一个23碱基对的序列称为终止子序列， 称为Tus的成分，它与DNA终止子序列结合。 是 建议研究Tus和Tus之间的蛋白质-DNA相互作用。 复制体 具体而言，建议： 1. 使用柱色谱法纯化和表征Tus蛋白 技术. 2. 研究野生型Tus蛋白与 染色体终止子序列，使用过滤器结合测定。 3. 系统地分析Tus结合至 通过产生位点特异性突变， 确定这些突变对结合的影响。 的相关性 结合亲和力和终止活性之间的关系也将是 确立了习 4. 产生突变Tus蛋白以定义DNA的活性位点 约束和终止。 5. 用遗传学和细胞生物学方法研究复制抑制的机制 生物化学方法