ACTIVATION AND REGULATION OF THE BGL OPERON IN E COLI

大肠杆菌 BGL 操纵子的激活和调控

• 批准号: 2392025
• 负责人: ANDREW WRIGHT
• 金额: $ 37.27万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392025
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; SDS polyacrylamide gel electrophoresis; bacterial DNA; bacterial genetics; binding proteins; dimer; gel mobility shift assay; gene expression; gene induction /repression; genetic regulation; genetic strain; mutant; nucleic acid sequence; operon; phosphorylation; protein kinase; site directed mutagenesis; tissue /cell culture; transcription factor; transcription termination

The long term goal of this project is to achieve a better understanding of how external signals are communicated to the machinery regulating gene expression in the cell. Our focus, with this goal in mind, has been a study of the bgl operon in E.coli, which is regulated by a two-component system consisting of a sensory protein, present in the cytoplasmic membrane and a regulatory protein, present in the cytoplasm. The sensor, termed BglF, is a bifunctional protein which transports substrate and which also acts as a protein kinase and phosphatase, regulating the activity of the regulator, BglG. In the absence of substrate, BglF phosphorylates BglG thus inactivating it, while in the presence of substrate, BglF dephosphorylates BglG, resulting in its activation. The active, non-phosphorylated form of BglG, is a dimer which binds to nascent RNA transcripts acting to prevent termination of transcription. During the next grant period we will define regions of contact between BglF and BglG that are essential for phosphorylation. This will be done by isolating and characterizing BglF mutants that fail to recognize and thus phosphorylate BglG. Such mutants will in turn be used to isolate BglG suppressors which will define the regions of contact between the two proteins. As part of this effort, we will identify the specific histidine that is phosphorylated in BglG. A major part of our effort will be a structure-function analysis of BglG. Residues crucial to RNA binding will be defined by site specific mutagenesis and also by selection of novel RNA sequences, produced by randomization of chosen segments of the RNA target. We will attempt to determine the crystal structure of the RNA binding domain bound to its target RNA. We will locate and characterize the sequences essential for BglG dimerization with the aim of understanding how phosphorylation blocks dimer formation. We will use in vitro approaches to reproduce the antitermination reaction in vitro. We wish to know whether BglG is sufficient for antitermination, if transcriptional pausing is essential for its action and precisely when the RNA target becomes accessible to the protein.

该项目的长期目标是更好地了解 外部信号如何传递至机械调节基因 细胞中的表达。考虑到这一目标，我们的重点是 对大肠杆菌中 bgl 操纵子的研究，该操纵子由两部分调节 由存在于细胞质中的感觉蛋白组成的系统 膜和调节蛋白，存在于细胞质中。传感器， 称为 BglF，是一种双功能蛋白，可转运底物和 它还充当蛋白激酶和磷酸酶，调节 调节器 BglG 的活性。在没有底物的情况下，BglF 磷酸化 BglG 从而使其失活，同时存在 底物，BglF 使 BglG 去磷酸化，导致其激活。这 BglG 的活性非磷酸化形式是一种二聚体，可结合新生 RNA 转录物的作用是防止转录终止。 在下一个资助期内，我们将定义之间的接触区域 BglF 和 BglG 对磷酸化至关重要。这将由 分离和表征无法识别的 BglF 突变体，从而 磷酸化 BglG。此类突变体将依次用于分离 BglG 抑制器将定义两者之间的接触区域 蛋白质。作为这项工作的一部分，我们将确定特定的组氨酸 BglG 被磷酸化。 我们工作的一个主要部分是 BglG 的结构功能分析。 对 RNA 结合至关重要的残基将通过位点特异性来定义 诱变以及通过选择新的RNA序列，产生的 RNA 靶标选定片段的随机化。我们将尝试 确定与其结合的RNA结合结构域的晶体结构 目标RNA。我们将定位并表征必要的序列 BglG 二聚化，旨在了解磷酸化如何阻断 二聚体形成。 我们将使用体外方法来重现抗终止反应 体外。我们想知道 BglG 是否足以抗终止， 转录暂停是否对其作用至关重要以及具体何时 RNA靶标变得可以被蛋白质所接近。