Single-molecule imaging of membrane-localized transcription complexes in bacteria

细菌膜定位转录复合物的单分子成像

• 批准号: 8284549
• 负责人: Julie Biteen
• 金额: $ 18.91万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8284549
• 关键词: Antibodies; Bacteria; Binding; Binding Sites; Biochemical; Cells; Chimeric Proteins; Chromosomes; Complex; Confocal Microscopy; Cytoplasm; DNA; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Elements; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; Image; Knowledge; Label; Life; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Outcome; Pathway interactions; Process; Proteins; Recruitment Activity; Regulatory Pathway; Research; Research Project Grants; Resolution; System; Techniques; Testing; Therapeutic; Transcription Coactivator; Transcription Regulatory Protein; Transcriptional Activation; Vibrio cholerae; Virulence; Work; cellular imaging; drug discovery; fitness; fluorescence imaging; imaging modality; interest; nanoscale; pathogen; promoter; research study; single molecule; tool; transcription factor; wasting

DESCRIPTION (provided by applicant): Transcription activation is typically carried out by soluble proteins engaging basal elements of the transcription apparatus - including promoter DNA and RNA polymerase - in the bacterial cytoplasm. In the Gram negative pathogen Vibrio cholerae, virulence gene expression is under control of an unusual set of membrane proteins. We hypothesize that a membrane complex including two activators, ToxR and TcpP, binds to the toxT promoter, recruits RNA polymerase, and activates toxT gene expression leading to activation of ToxT-controlled virulence genes. The mechanism by which membrane proteins can access DNA in the cell and recruit RNA polymerase has not been uncovered with standard genetic and biochemical approaches. Single-molecule imaging methods with nanometer-scale resolution now make it possible to investigate this mechanism in living cells, and these techniques will be applied to the ToxR/TcpP system to test specific hypotheses. This exploratory proposal has the following two specific aims: 1. Construct Vibrio cholerae strains expressing photo-activatable fluorescent fusion proteins of ToxR and TcpP, and mark toxT promoter DNA in the V. cholerae genome using the lacO operator site for binding of a LacI-EYFP fusion protein. 2. Carry out single-molecule super-resolution imaging in live cells to test specific hypotheses about the mechanism and dynamics by which membrane activators bind to toxT promoter DNA for activation of virulence gene expression. PUBLIC HEALTH RELEVANCE: Bacteria control gene expression tightly so as not to waste energy and thereby decrease their fitness. These mechanisms represent good targets for drug discovery to control bacterial pathogens. This work will examine single molecules in living cells using state of the art approaches, thereby uncovering knowledge that is the most suitable for eventual therapeutic discovery research.

描述（由申请人提供）：转录激活通常是通过可溶性蛋白质与细菌细胞质中转录装置的基础元件（包括启动子 DNA 和 RNA 聚合酶）接合来进行的。在革兰氏阴性病原体霍乱弧菌中，毒力基因表达受到一组不寻常的膜蛋白的控制。我们假设包含两种激活剂 ToxR 和 TcpP 的膜复合物与 toxT 启动子结合，招募 RNA 聚合酶，并激活 toxT 基因表达，从而激活 ToxT 控制的毒力基因。标准遗传和生化方法尚未揭示膜蛋白接触细胞中 DNA 并招募 RNA 聚合酶的机制。具有纳米级分辨率的单分子成像方法现在使得在活细胞中研究这种机制成为可能，这些技术将应用于ToxR/TcpP系统来测试特定的假设。该探索性提案有以下两个具体目标： 1. 构建表达 ToxR 和 TcpP 光激活荧光融合蛋白的霍乱弧菌菌株，并使用 lacO 操纵位点在霍乱弧菌基因组中标记 toxT 启动子 DNA，以结合 LacI- EYFP融合蛋白。 2. 在活细胞中进行单分子超分辨率成像，以测试有关膜激活剂与 toxT 启动子 DNA 结合以激活毒力基因表达的机制和动力学的具体假设。 公共健康相关性：细菌严格控制基因表达，以免浪费能量，从而降低其健康水平。这些机制代表了控制细菌病原体药物发现的良好靶标。这项工作将使用最先进的方法检查活细胞中的单分子，从而揭示最适合最终治疗发现研究的知识。