PHYSICAL CHEMISTRY OF NUCLEIC ACIDS

在这一应用中，我们提出了各种互补性的单分子分析来研究机械 转录延伸过程中单个大肠杆菌RNA聚合酶分子的移位。通过这些 研究中，我们试图确定RNA聚合酶(RNAP)在沿 模板和表征核苷酸加成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力化学循环的细节。具体来说，我们建议： 1.在连续、不间断的延伸过程中使用集成的 目前在我们实验室运行的光学捕捉/流控视频显微镜。 2.描述电机的机械力化学循环。为此，我们将获得力-速度曲线 大肠杆菌在各种条件下的RNA聚合酶。这些曲线将使用分子马达进行分析。 理论是我们实验室最近发展起来的。 3.建立了直接观察RNAP易位动力学的高分辨率易位分析方法 在单BP分辨率下。 4.通过单分子实验表征RNAP在扭转应力作用下的移位动力学 在大肠杆菌旋转酶存在和不存在的情况下，用扭曲限制的DNA进行转录分析。 通过这里描述的单分子研究，将有可能跟踪单个分子事件 否则就会在总体测量的总体平均值中被忽略。这些事件，涉及到动力学 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，机械力将被用作一种新的可控性 变量来表征这种马达酶中的机械力化学转导过程。

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.