Transcription-Coupled DNA Supercoiling

描述(申请人提供)：几十年来，人们已经认识到，通过RNA聚合酶沿DNA双螺旋转录可以增强局部DNA超螺旋。这个过程被一个“双超螺旋结构域”模型很好地解释了，在这个模型中，正的DNA超螺旋在转位的RNA聚合酶之前产生，负的超螺旋在其后面产生。然而，分子水平上的详细机制仍不清楚。例如，DNA拓扑障碍如何影响转录偶联DNA超卷曲(TCDS)还没有完全了解。这项研究的长期目标是了解TCDS和DNA拓扑障碍的分子机制和生物学功能。这项应用的目的是确定是什么性质使某些DNA-环和包裹蛋白发挥拓扑屏障和阻止超螺旋扩散的作用，研究DNA拓扑屏障如何调节大肠杆菌细胞中的TCDS，以及检测定位的瞬时TCDS如何激活体内的基因表达。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，在大肠杆菌细胞中，细胞使用基于核蛋白的拓扑屏障将TCDS限制在局部区域，从而极大地影响附近的耦合DNA交易。我们的假设是基于我们实验室提供的强大的初步数据，并将通过追求以下三个具体目标进行验证：1)确定DNA拓扑屏障的分子机制，并研究DNA拓扑屏障如何将超螺旋限制在局部区域并影响TCDS的效率；2)检测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中的转录，以及DNA拓扑屏障如何影响TCDS激活转录的效率。这些实验提供的信息最终将使我们更好地了解TCDS的机制，特别是DNA拓扑障碍在TCDS中的作用以及基因转录和表达的耦合。

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.