PROTEIN/DNA INTERACTION AT THE PHEBA PROMOTER

PHEBA 启动子处的蛋白质/DNA 相互作用

• 批准号: 2292139
• 负责人: Ananda Mohan Chakrabarty
• 金额: $ 2.51万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-03-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2292139
• 关键词: DNA footprinting; SDS polyacrylamide gel electrophoresis; bacterial DNA; bacterial proteins; gel mobility shift assay; gene induction /repression; gene mutation; genetic promoter element; microorganism metabolism; mutant; nucleic acid sequence; operon; site directed mutagenesis

Many microorganisms metabolize aromatic compounds via formation of catechol as an intermediate by a pathway called the ortho pathway. In Pseudomonas putida, the catechol degradative genes catB and catC are clustered together and organized as an operon. The catBC operon is regulated by a positive regulator CatR which binds upstream of the catB gene, and in presence of the inducer cis,cis-muconate (CCM), binds to two specific sites called repression binding site (RBS) and activation binding site (ABS). Binding at the RBS in absence of the inducer allows autoregulation by CatR of its own gene (catR) which is transcribed divergently from the catBC promoter, but does not lead to activation of the catBC operon. In presence of the inducer CcM, CatR undergoes a conformational change that allows it to bind the additional sequence ABS which leads to transcription initiation at the catBC promoter. Many microorganisms metabolize the toxic compound phenol via formation of catechol as an intermediate. However, in such cases, the catechol derived from phenol is metabolized by a different pathway called the meta pathway. In various well-studied systems, the meta pathway genes are also clustered to form an operon which is regulated by a positive regulator having homology to response regulators of the two-component system. Dr. Kivisaar's group has isolated a strain of P. putida that metabolizes phenol via catechol as an intermediate where the catechol, unlike other well-studied phenol pathways, undergoes mineralization via the chromosomally encoded ortho pathway. The evolved genes pheBA, encoding phenol hydroxylase (pheA gene product) and catechol l,2-dioxygenase (pheB gene product), have a promoter that resembles catBC promoter and allows binding by CatR for functional activation of the pheBA genes. Thus we propose to study comparative interactions of CatR with catBC and pheBA promoters to understand how and why evolution took a short-cut to simply evolve a couple of genes for phenol degradation, instead of a whole pathway that normally evolves in natural microorganisms.

许多微生物通过形成芳香族化合物代谢芳香族化合物。 邻苯二酚作为中间体通过一种叫做邻位途径的途径。 在 在恶臭假单胞菌中，邻苯二酚降解基因catB和catC是 聚集在一起形成操纵子 catBC操纵子是 由结合catB上游的正调节因子CatR调节 基因，并在诱导剂顺式，顺式-粘康酸（CCM）的存在下，结合两个 特异性位点称为阻遏结合位点（RBS）和激活结合位点 地点（ABS）。 在不存在诱导物的情况下在RBS处的结合允许 通过自身基因（catR）的CatR自动调节， 从catBC启动子发散，但不导致 catBC操纵子。 在诱导剂CcM的存在下，CatR经历一个 允许其结合额外序列ABS的构象变化 这导致在catBC启动子处的转录起始。许多 微生物通过形成 儿茶酚作为中间体。 然而，在这种情况下，儿茶酚衍生物 通过一种不同的途径代谢，称为Meta途径。 在各种研究充分的系统中，Meta途径基因也成簇 以形成由正调节子调节的操纵子，所述正调节子具有 与双组分系统的反应调节子同源。博士 Kivisaar的研究小组已经分离出一种恶臭假单胞菌， 苯酚通过邻苯二酚作为中间体，其中邻苯二酚，不同于其他 经过充分研究的苯酚途径， 染色体编码的邻位途径。 进化的基因pheBA，编码 酚羟化酶（pheA基因产物）和邻苯二酚1，2-双加氧酶（pheB 基因产物），具有类似于catBC启动子的启动子并允许 通过CatR结合以功能性激活pheBA基因。 因此我们 建议研究CatR与catBC和pheBA的比较相互作用 推动者了解进化是如何以及为什么走捷径， 进化出几个降解苯酚的基因，而不是一个完整的 通常在自然微生物中进化的途径。