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

描述(申请人提供)：转录激活通常是由与细菌细胞质中转录装置的基本元素--包括启动子DNA和RNA聚合酶--结合的可溶性蛋白进行的。在革兰氏阴性霍乱弧菌中，毒力基因的表达受到一组不同寻常的膜蛋白的控制。我们推测，包含两个激活剂ToxR和TCPP的膜复合体与ToxT启动子结合，招募RNA聚合酶，激活ToxT基因的表达，从而激活ToxT控制的毒力基因。用标准的遗传和生化方法还没有发现膜蛋白可以访问细胞中的DNA并招募RNA聚合酶的机制。现在，具有纳米级分辨率的单分子成像方法使得在活细胞中研究这一机制成为可能，这些技术将被应用于ToxR/TCPP系统，以检验特定的假设。本探索性方案有以下两个具体目的：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，并利用LACO操纵子结合LacI-EYFP融合蛋白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成像，以验证关于膜激活剂与oxT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。

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.