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

描述（由申请人提供）：转录激活通常通过可溶性蛋白质接合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）来进行。在革兰氏阴性病原体霍乱弧菌中，毒力基因的表达受一组不寻常的膜蛋白的控制。我们假设，膜复合物，包括两个激活剂，ToxR和TcpP，结合到toxT启动子，招募RNA聚合酶，并激活toxT基因的表达，导致激活的ToxT控制的毒力基因。膜蛋白可以进入细胞中的DNA并募集RNA聚合酶的机制尚未用标准的遗传和生物化学方法揭示。具有纳米级分辨率的单分子成像方法现在可以在活细胞中研究这种机制，这些技术将应用于ToxR/TcpP系统以测试特定的假设。这一探索性建议有以下两个具体目标：1.构建表达ToxR和TcpP的光激活荧光融合蛋白的霍乱弧菌菌株，并使用LacI-EYFP融合蛋白结合的lacO操纵位点标记霍乱弧菌基因组中的toxT启动子DNA。2.在活细胞中进行单分子超分辨率成像，以测试关于膜激活剂与toxT启动子DNA结合以激活毒力基因表达的机制和动力学的特定假设。

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.