TRANSCRIPTION STUDIED AT THE MOLECULAR LEVEL

分子水平的转录研究

• 批准号: 6180966
• 负责人: STEVEN M BLOCK
• 金额: $ 25.37万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180966
• 关键词: DNA directed RNA polymerase; Escherichia coli; enzyme activity; enzyme mechanism; genetic transcription; molecular genetics; nanotechnology; optics; technology /technique

This application describes a research program to characterize the process of transcription by E. Coli RNA polymerase molecules at the single molecule level by means of novel biophysical methods of optical trapping and nanometer-level position sensing. The ultimate objective of this proposal is to investigate the mechanochemical properties of this processive enzyme, to gain an understanding of how the DNA-direct RNA synthesis is coupled to translocation along the DNA template. The specific aims of this proposal are: 1) To determine the force- velocity relationship for RNAP and characterize themechanochemical properties of this motor protein. These studies will investigate the mechanochemical behavior of this enzyme spanning the full range of external load forces; i.e., below, at and above the stall force of the motor. Similarly, the force- velocity relationship of the enzyme working in the presence of an external load applied in the direction of the motion will be characterized. 2) Investigate transcriptional pausing, stalling and arrest by RNAP and its relationship to the nature of the DNA template. These experiments will investigate whether the arrest of the enzyme is a probabilistic or a deterministic phenomenon, whether it is dictated by the sequence of the template, and if reversible and irreversible arrests are influenced by the present and the magnitude of the external load. The sequence effects onpausing and stalling will be studied on complex sequences, on direct repeat templates, and on homopolymers. Attempts will be also made to observe, directly, slippage under the external load. Finally, these studies will investigate what is the effect of transcript cleavage factors such as GreA and GreB on pausing and stalling. 3) Study the fluctuation behavior of the dynamics of individual transcribing enzymes. A variance analysis will be applied to obtain information about the number and duration of the rate-limiging steps in the enzyme reaction, as a way to get an insight into the molecular mechanism responsible for the mechanochemical coupling in RNAP. 4) Studies of the fine structure of the translocation of transcribing single RNAP molecules. These studies will be designed to I) investigate the effect of torsional strain during transcription, ii) obtain direct evidence of discontinuous or "inch worming" movement of the polymerase along the template, particularly during the early phases of transcription, and iii) attempt to resolve the process of transcription to single base pair resolution.

该应用程序描述了一个研究计划，以表征 大肠杆菌RNA聚合酶分子的转录过程 通过新颖的生物物理方法的单分子水平的水平 光学诱捕和纳米级位置感测。 这 该提议的最终目标是调查 该过程酶的机械化学特性，以获得 了解如何将DNA直接RNA合成耦合到 沿着DNA模板易位。 该提案的具体目的是：1）确定力 - RNAP和特征的速度关系 该运动蛋白的主级化学特性。 这些 研究将研究此的机械化学行为 跨越全部外部载荷力的酶；即，下面， 在电动机的摊位上和之上。 同样，力 在存在的情况下工作的酶的速度关系 朝着运动方向施加的外部负载将是 特征。 2）调查转录暂停，失速和 RNAP及其与DNA性质的关系 模板。 这些实验将调查是否逮捕 酶是概率或确定性现象， 它是否由模板的序列决定，以及是否由 可逆和不可逆转的逮捕受到当前的影响 以及外部负载的大小。 序列效应 将在复杂序列上研究和失速 直接重复模板和均聚物。 尝试将是 还可以直接观察外部负载下的滑倒。 最后，这些研究将研究转录的影响 裂解因素（例如Grea和Greb）在暂停和失速方面。 3）研究个体动力学的波动行为 转录酶。 将应用方差分析以获得 有关限制速率的数量和持续时间的信息 酶反应的一步，以了解 负责机械化学的分子机制 耦合在rnap中。 4）研究 转录单RNAP分子的易位。 这些 研究将设计为i）研究扭转的影响 转录期间的菌株，ii）获得直接证据 聚合酶的不连续或“英寸蠕动”运动 沿模板，尤其是在早期的早期阶段 转录和iii）试图解决的过程 转录单基对分辨率。