MECHANISM OF GENE REGULATION BY A LYSR REPRESSOR

这份提案描述了一个为期两年的调查财产的项目 假单胞菌中新发现的原核抑制因子DgdR 洋葱。DgdR是由839个碱基的dgdR基因编码的，它与 它正好领先于2，2-二烷基甘氨酸的结构基因DGDA 脱羧酶。正如我们和其他人通过序列比对所表明的那样，DgdR 是DNA结合蛋白LysR家族的成员。长距离的 该项目的目标是在结构和机械方面进行解释 为什么，尽管它们有相似的序列，这种蛋白质是一种 转录抑制因子，而其余的LysR蛋白只有一个 是激活剂。我们已经从凝胶迁移率变化分析中获得了证据 使用部分纯化的阻遏物制剂和DNA片段 包括DGDA和DGDR的5‘端，即(I)阻遏因子可形成 在两个操作符之间循环，一个350个核苷酸进入DGDA基因，以及 另外200个核苷酸在上游，以及(Ii)该环在体外被打破 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，该模型包括DGD系统的形成 二聚体-DNA、四聚体-DNA和环状四聚体-DNA复合体。我们建议 利用重组DNA首次大量纯化阻遏蛋白 技术，然后重复我们初步的凝胶迁移率变化实验 使用纯化的蛋白质。有无DNase I足迹 各种氨基酸将提供对循环公设的直接测试。 此外，抑制子的氨基酸的形状和表面性质 将使用二烷基甘氨酸类似物来探测结合部位，这些类似物与 α取代基的大小和疏水性。五花八门 之前在本实验室合成的多氟二烷基甘氨酸将是 在体内和体外被测试为潜在的诱导剂，以及胺和 模拟已知的二烷基甘氨酸其他部分的羧酸 诱导者。拟议的研究将提供关于 基本的遗传控制机制可能广泛存在于 原核生物，因此很可能直接参与了许多细菌 疾病过程。

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.