TERMINATION OF DNA REPLICATION IN ESCHERICHIA COLI

本提案中提出的实验旨在提供一种 更好地理解DNA终止的生化基础 在大肠杆菌中复制。大肠杆菌的两种成分 已经确定了终止系统：DNA组件，它包括 一个称为终止子序列的23个碱基对的序列和一个蛋白质 称为Tus的成分，它与DNA终止子序列结合。它是 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，建议的目标是： 1.用柱层析法纯化和鉴定Tus蛋白 技巧。 2.研究野生型Tus蛋白与Tus蛋白的结合作用 使用滤膜结合分析的染色体终止子序列。 3.系统分析TUS与TUS结合的序列要求 终止子序列通过产生位点特异性突变和 确定这些突变对结合的影响。一种相关性 结合亲和力和终止活动之间的关系也将 已经成立了。 4.产生突变型Tus蛋白以确定DNA的活性部位 绑定和终止。 5.从基因和分子水平研究复制抑制的机制 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.