Transcription-Coupled DNA Supercoiling

描述（由申请人提供）：几十年来，人们已经认识到，RNA聚合酶沿着DNA双螺旋转录可以增强局部DNA超卷曲。这个过程被一个“双超螺旋结构域”模型优雅地解释了，在这个模型中，正DNA超螺旋在易位RNA聚合酶前面产生，负DNA超螺旋在它后面产生。然而，在分子水平上的详细机制仍然不可用。例如，DNA拓扑屏障如何影响转录偶联DNA超卷曲（TCDS）尚未完全了解。本研究的长期目标是了解TCDS和DNA拓扑屏障的分子机制和生物学功能。本应用程序的目的是确定哪些特性使某些DNA环和包裹蛋白作为拓扑屏障起作用并阻止超线圈扩散，研究DNA拓扑屏障如何调节大肠杆菌细胞中的TCDS，并检查局部瞬时TCDS如何激活体内基因表达。我们的中心假设是，RNA聚合酶的TCDS是大肠杆菌细胞中主要的染色体重塑力量，其中细胞使用基于核蛋白的拓扑屏障将TCDS限制在局部区域，因此极大地影响了附近的偶联DNA交易。我们的假设是在我们实验室产生的强有力的初步数据的基础上制定的，并将通过以下三个具体目标进行测试：1)确定DNA拓扑屏障的分子机制，并研究DNA拓扑屏障如何将超级线圈限制在局部区域并影响TCDS的效率；2)研究DNA拓扑屏障在大肠杆菌细胞TCDS中的作用；3)研究瞬时、动态的TCDS如何激活大肠杆菌细胞的转录，以及DNA拓扑屏障如何影响TCDS激活转录的效率。这些实验的结果将有助于我们更好地理解TCDS的机制，特别是DNA拓扑屏障在TCDS中的作用以及耦合基因的转录和表达。

DESCRIPTION (provided by applicant): For decades, it has been recognized that transcribing along the DNA double helix by a RNA polymerase can enhance localized DNA supercoiling. This process has been elegantly explained by a "twin-supercoiled- domain" model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase and negative supercoils behind it. However, detailed mechanisms at the molecular level are still unavailable. For instance, how DNA topological barriers affect transcription-coupled DNA supercoiling (TCDS) has not been fully understood. The long-term goal of the proposed research is to understand the molecular mechanisms and biological functions of TCDS and DNA topological barriers. The objectives of this application are to determine what properties make certain DNA-looping and -wrapping proteins function as topological barriers and block supercoil diffusion, to study how DNA topological barriers regulate TCDS in E. coli cells, and to examine how localized, transient TCDS activates gene expression in vivo. Our central hypothesis is that TCDS by RNA polymerases is a major chromosome remodeling force in E. coli cells where the cells use nucleoprotein-based, topological barriers to confine TCDS to localized regions and, as a result, greatly influence the nearby, coupled DNA transactions. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory and will be tested by pursuing the following three specific aims: 1) to determine molecular mechanisms of DNA topological barriers and examine how the DNA topological barriers confine supercoils to localized regions and affect the efficiency of TCDS; 2) to examine the role of DNA topological barriers in TCDS in E. coli cells; 3) to investigate how transient, dynamic TCDS activates transcription in E. coli cells and how DNA topological barriers affect the efficiency of transcription activation by TCDS. Information from the proposed experiments will ultimately provide us with a better understanding of the mechanisms of TCDS, especially the roles of DNA topological barriers in TCDS and the coupled gene transcription and expression.