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理解 如何将外部信号传达给调节基因的机械 在细胞中的表达。考虑到这个目标，我们的重点一直是 E.COLI中BGL操纵子的研究，该操纵子受两个组件调节 由感官蛋白组成的系统，存在于细胞质中 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，可传输底物和 它也充当蛋白激酶和磷酸酶，调节 调节器的活性BGLG。在没有底物的情况下，BGLF 因此，磷酸化bglg因此使它失活，而在存在的情况下 底物BGLF去磷酸化bglg，导致其激活。这 活性，非磷酸化形式的BGLG是一个二聚体，与新生 RNA转录本作用以防止转录终止。 在下一个赠款期间，我们将定义 BGLF和BGLG对于磷酸化至关重要。这将由 隔离和表征无法识别的BGLF突变体，从而 磷酸化BGLG。这样的突变体将又用于隔离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 在BGLG中磷酸化。 我们努力的主要部分是BGLG的结构 - 功能分析。 对RNA结合至关重要的残基将由位点特异性定义 诱变，也是通过选择新的RNA序列的选择，由 RNA靶标的所选段的随机化。我们将尝试 确定RNA结合结构域结合的晶体结构 靶RNA。我们将找到并表征对 BGLG二聚化的目的是了解磷酸化如何阻止 二聚体形成。 我们将使用体外方法重现抗授权反应 体外。我们希望知道BGLG是否足以进行抗授权， 如果转录暂停对其行动至关重要，并且恰恰是 RNA靶标可以访问蛋白质。