PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

在这个应用中，我们提出了各种互补的单分子分析来研究在转录延伸过程中单个大肠杆菌RNA聚合酶分子的机械易位。通过这些研究，我们试图确定RNA聚合酶（RNAP）沿着模板移动时的复杂动力学，并表征核苷酸添加和易位之间的关系，作为最终以高分辨率解剖其机械化学循环细节的第一步。具体而言，我们建议：1。在持续的、不间断的延伸过程中，使用集成光学捕获/流量控制视频显微镜来表征暂停和停止行为。2. 描述电机的机械化学循环。为此，我们将得到大肠杆菌RNA聚合酶在各种条件下的力-速度曲线。这些曲线将使用我们实验室最近开发的分子马达理论进行分析。3. 建立一个高分辨率的易位实验，在单bp分辨率下直接观察RNAP易位的动态。4. 在大肠杆菌扭转酶存在和不存在的情况下，通过使用扭转约束DNA进行单分子转录测定，表征RNAP在扭转应力下的易位动力学。通过这里描述的单分子研究，将有可能跟踪单个分子事件，否则这些事件将在总体测量的总体平均中被遗漏。这些事件涉及酶的结构和功能的动态变化，是基因表达调节过程的核心。最后，在这里描述的实验中，机械力将作为一个新的可控变量来表征这种运动酶的机械化学转导过程。

In this application we propose various complementary single molecule assays to investigate the mechanical translocation of individual E. coli RNA polymerase molecules during transcription elongation. Through these studies, we seek to determine the complex dynamics of RNA polymerase (RNAP) as it moves along the template and characterize the relationship between nucleotide addition and translocation as a first step to ultimately dissect the details of its mechanochemical cycle at high resolution. Specifically, we propose to: 1. Characterize pausing and arrest behavior during continued, uninterrupted elongation using an integrated optical trapping/flow control video microscope currently operating in our laboratory. 2. Characterize the mechanochemical cycle of the motor. To this end, we will obtain force-velocity curves for E. coli RNA polymerase under a variety of conditions. These curves will be analyzed using a molecular motor theory recently developed in our laboratory. 3. Develop a high resolution translocation assay to directly observe the dynamics of translocation of RNAP at single bp resolution. 4. Characterize the dynamics of translocation of RNAP against torsional stress by performing single molecule transcription assays with torsionally constrained DNA both in the presence and absence of E. coli gyrase. Through the single molecule studies described here, it will be possible to follow individual molecular events that would otherwise be missed in the ensemble average of bulk measurements. These events, involving dynamical changes in structure and function of the enzyme, lie at the heart of the process of regulation of gene expression. Finally, in the experiments described here, mechanical force will be used as a new controllable variable to characterize the process of mechanochemical transduction in this motor enzyme.