PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

在本申请中，我们提出了各种互补的单分子测定来研究机械的 个体E.在转录延伸过程中的RNA聚合酶分子。通过这些 研究，我们试图确定RNA聚合酶（RNAP）的复杂动力学，因为它沿着沿着 模板化并表征核苷酸添加和易位之间的关系作为第一步， 最终以高分辨率剖析其机械化学循环的细节。具体而言，我们建议： 1.使用集成的 光学捕获/流控视频显微镜目前在我们的实验室运行。 2.描述发动机的机械化学循环。为此，我们将获得力-速度曲线， E. coli RNA聚合酶在多种条件下的活性。这些曲线将使用分子马达进行分析 我们实验室最近开发的理论。 3.建立一种高分辨率的直接观察RNAP易位动力学的方法 在单个BP分辨率下。 4.通过进行单分子实验，表征RNAP对抗扭转应力的移位动力学 在存在和不存在大肠杆菌促旋酶的情况下，用扭转约束的DNA进行转录测定。 通过这里描述的单分子研究，将有可能跟踪单个分子事件， 否则将在整体测量的总体平均中被遗漏。这些事件，涉及动力学 酶的结构和功能的变化，是基因调控过程的核心。 表情最后，在这里描述的实验中，机械力将被用作新的可控 变量来表征该马达酶中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 mechanochemicalcycle 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-velocitycurves 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 RNAPagainst 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 describe here, it will be possible to follow individual molecular events that would otherwise be missed in the ensemble average ofbulk 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.