MECHANISM OF GENE REGULATION BY A LYSR REPRESSOR

LYSR 阻遏物的基因调控机制

• 批准号: 2187004
• 负责人: JOHN W KELLER
• 金额: $ 9.59万
• 依托单位: UNIVERSITY OF ALASKA FAIRBANKS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-06-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187004
• 关键词: DNA binding protein; DNA footprinting; Pseudomonas; alanine; amines; bacterial genetics; carboxylate; glycine; mutant; prokaryote; protein sequence; protein structure function; recombinant DNA; regulatory gene; transcription factor

This proposal describes a two-year project to investigate the properties of DgdR, a newly discovered prokaryotic repressor from Pseudomonas cepacia. DgdR is encoded by the 839-bp dgdR gene, which diverges from and is just ahead of dgdA, the structural gene of a 2,2-dialkylglycine decarboxylase. As we and others have shown by sequence alignment, DgdR is a member of the LysR family of DNA binding proteins. The long range objective of the project is to explain in structural and mechanistic terms why, although they have similar sequences, this protein is a repressor of transcription, while the rest of the LysR proteins but one are activators. We have obtained evidence from gel mobility shift assays using a partially purified repressor preparation and DNA segments including the 5'ends of dgdA and dgdR that (i) the repressor may form a loop between two operators, one 350 nucleotides into the dgdA gene, and the other 200 nucleotides upstream, and (ii) the loop is broken in vitro by 2-methylalanine, an amino acid that induces dgdA expression in vivo. A model is proposed for the dgd system that includes formation of dimer-DNA, tetramer-DNA, and looped tetramer-DNA complexes. We propose to first purify the repressor protein in quantity using recombinant DNA techniques, then repeat our preliminary gel mobility shift experiments using the purified protein. DNase I footprinting with and without various amino acids will provide a direct test of the looping postulate. Also, the shape and surface properties of the repressor's amino acid binding site will be probed using dialkylglycine analogs that differ by the size and hydrophobicity of the alpha substituents. Various polyfluoro dialkylglycines previously synthesized in this lab will be tested in vivo and in vitro as potential inducers, as well as amines and carboxylic acids that mimic other portions of the known dialkylglycine inducers. The proposed research will provide new information about a fundamental genetic control mechanism that is probably widespread in prokaryotes, and thus is likely to be directly involved in many bacterial disease processes.

该提案描述了一个为期两年的项目， DgdR是一个新发现的假单胞菌原核阻遏蛋白 洋葱。 DgdR由839-bp的dgdR基因编码，该基因与 并且刚好在dgdA（2，2-二烷基甘氨酸的结构基因）之前 脱羧酶 正如我们和其他人通过序列比对所示，DgdR 是DNA结合蛋白的LysR家族的成员。 远程 该项目的目的是解释在结构和机械 术语为什么，虽然它们有相似的序列，这种蛋白质是一种 转录阻遏物，而其余的LysR蛋白，但一个 是激活剂。 我们已经从凝胶迁移率变动分析中获得了证据 使用部分纯化的阻遏物制备物和DNA片段 包括dgdA和dgdR的5 '末端，（i）阻遏物可以形成一个 两个操纵子之间的环，一个350个核苷酸进入dgdA基因， 上游的另外200个核苷酸，和（ii）环在体外断裂 通过2-甲基丙氨酸，一种在体内诱导dgdA表达的氨基酸。 提出了一种dgd系统的模型， 二聚体-DNA、四聚体-DNA和环状四聚体-DNA复合物。 我们提出 首先使用重组DNA大量纯化阻遏蛋白 技术，然后重复我们的初步凝胶迁移率变化实验 使用纯化的蛋白质。 有和无DNA酶I足迹 各种氨基酸将提供对循环假设的直接检验。 同样，阻遏蛋白氨基酸的形状和表面性质 将使用二烷基甘氨酸类似物探测结合位点， α取代基的大小和疏水性。 各种 本实验室先前合成的多氟二烷基甘氨酸将 在体内和体外测试作为潜在的诱导剂，以及胺和 模拟已知二烷基甘氨酸的其它部分的羧酸 诱导剂。 这项研究将提供关于一个新的信息。 一种基本的遗传控制机制， 原核生物，因此可能直接参与许多细菌 疾病过程。