PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

超出提供的空间。在这一应用中，我们提出了各种互补性的单分子分析方法来研究单个大肠杆菌RNA聚合酶分子在转录延伸过程中的机械移位。通过这些研究，我们试图确定RNA聚合酶(RNAP)沿模板移动的复杂动力学，并表征核苷酸加成和易位之间的关系，作为最终以高分辨率剖析其机械力化学循环的细节的第一步。具体地说，我们建议：1.使用我们实验室目前运行的集成光学捕捉/流动控制视频显微镜来表征连续、不间断延伸过程中的暂停和停止行为。2.描述电机的机械力化学循环。为此，我们将获得不同条件下大肠杆菌RNA聚合酶的力-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.建立了一种高分辨率的RNAP易位分析方法，可以直接观察单个BP分辨率下RNAP的易位动力学。4.在大肠杆菌旋转酶存在和不存在的情况下，用扭转限制的DNA进行单分子转录分析，以表征RNAP在扭转应力下的转位动力学。通过这里描述的单分子研究，将有可能跟踪单个分子事件，否则这些事件将被整体测量的整体平均所遗漏。这些事件涉及酶的结构和功能的动态变化，位于基因表达调控过程的核心。最后，在本文描述的实验中，机械力将作为一个新的可控变量来表征该马达酶的机械力化学转导过程。表演网站========================================Section End===========================================

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===========================================