TERMINATION OF DNA REPLICATION IN ESCHERICHIA COLI

本提案中提出的实验旨在提供 更好地了解 DNA 终止的生化基础 在大肠杆菌中复制。 大肠杆菌的两个组成部分 已确定终止系统：DNA 成分，其中包括 称为终止子序列的 23 个碱基对序列和蛋白质 称为 Tus 的成分，它与 DNA 终止子序列结合。 它是 提议研究 Tus 和 复制体。 具体来说，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蛋白与野生型Tus蛋白之间的结合相互作用 使用过滤结合测定的染色体终止子序列。 3. 系统分析Tus与结合的序列要求 通过产生位点特异性突变来终止子序列和 确定这些突变对结合的影响。 相关性 结合亲和力和终止活性之间也将是 成立。 4. 生成突变 Tus 蛋白以定义 DNA 的活性位点 绑定和终止。 5. 利用基因和技术研究复制抑制机制 生化方法。

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.