PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

超出所提供的空间。 在本申请中，我们提出了各种互补的单分子测定来研究单个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移动，并表征核苷酸添加和易位之间的关系作为第一步，最终剖析其mechanochemicalcycle在高分辨率的细节。具体而言，我们建议：1.表征暂停和逮捕行为在持续，不间断的伸长使用集成的光学捕获/流量控制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，我们将得到E. coli RNA聚合酶在多种条件下的活性。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.开发一种高分辨率的易位测定法，以在单个bp分辨率下直接观察RNAP易位的动力学。4.通过在存在和不存在大肠杆菌促旋酶的情况下使用扭转约束DNA进行单分子转录测定，表征RNAP针对扭转应力的易位动力学。通过这里描述的单分子研究，将有可能跟踪个体分子事件，否则这些事件将在整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，是基因表达调控过程的核心。最后，在这里描述的实验中，机械力将被用作一个新的可控变量来表征在这个马达酶的机械化学转导过程。性能现场=

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================