TRANSIENT STATE KINETICS OF TRANSCRIPTION ELONGATION

描述：本提案中提出的研究旨在 阐明了E.大肠杆菌RNA 聚合酶催化新生RNA转录物的合成。 的 这种努力的理由在于选择E.大肠杆菌RNA聚合酶 作为一类酶的最好理解的成员， 以非解离方式处理RNA转录物的产生。 因此，E.大肠杆菌酶作为一个原型， 复杂的真核RNA聚合酶。 酶的功能是 在DNA模板的指导下， 一条新生的RNA链，具有极高的保真度， 有效的营业额。 聚合酶沿着DNA模板的运动 然而，时间是不均匀的，伊利博士将其定义为给定时间的停留时间。 模板位置，其范围可以从10 ms到几秒。 之一 该提案的主要目标是定义序列依赖 聚合酶沿着DNA模板的传输速率的差异 并确定特定序列在控制速率中的作用 聚合酶聚合。 这表明，序列依赖 导致停顿的转录速率可能是 一个监管网络，在控制 基因在转录水平上的表达。 因此，建议 开始了一个雄心勃勃的项目，以确定和动力学定义所有 转录途径中的步骤，并确定哪些可能是 利率限制，因此受到监管，为正确和 不正确的核苷酸掺入和切除。 的方法 程序集中于个体的瞬态动力学研究 聚合酶循环中的反应步骤。 自从RNA聚合酶出现以来 是一种多功能酶， RNA转录物的外切核酸裂解，这是必不可少的， 确定核苷酸掺入的限速步骤， 切除并确定序列元件如何改变这些步骤。 待研究的具体步骤包括核苷酸的动力学 与定义速率限制步骤相关的问题合并 在正确的核苷酸掺入中， 转录本位置、序列和长度对正确和 不正确的核苷酸掺入，这些动力学步骤的联系 整体转录保真度的表现， 聚合酶切除核苷酸的步骤， 转录物切割和焦磷酸解之间的分配 核苷酸切除，以及转录物位置的影响， 序列和长度对核苷酸切除动力学的影响。

DESCRIPTION: The studies presented in this proposal are designed to elucidate the detailed kinetic mechanisms by which E. coii RNA polymerase catalyzes the synthesis of the nascent RNA transcript. The rationale for this effort lies in the choice of E. coli RNA polymerase as the best understood member of the class of enzymes which bind and process in a non-dissociative manner the production of RNA transcripts. As such, the E. coli enzyme serves as a prototype for the more complicated eukaryotic RNA polymerases. The enzyme functions to synthesize, under the direction of the DNA template, the formation of a nascent RNA chain with exceedingly high fidelity with catalytical effient turnover. The motion of the polymerase along the DNA template is uneven, however, and Dr. Erie defines this as a dwell time at a given template position which can range from 10 ms to several seconds. One of the main goals of the proposal is to define the sequence dependent differences in the transit rate of the polymerase along the DNA template and to determine the role of specific sequences in controlling the rate of polymerase polymerization. It is suggested that sequence-dependent transcription rates which result in pausing may be central features of a regulatory network, which plays an important role in the control of gene expression at the transcription level. As such, the proposal embarks on an ambitious project to identify and kinetically define all the steps in the transcription pathway and to determine which might be rate-limiting, and hence subject to regulation, for both correct and incorrect nucleotide incorporation and excision. The method of procedure centers on transient-state kinetic studies of the individual reaction steps in the polymerase cycle. Since RNA polymerase appears to be a multifunctional enzyme that catalyzes the endo- and exonucleolytic cleavage of the RNA transcript, it is essential to identify the rate-limiting steps to nucleotide incorporation and excision and to determine how sequence elements alter these steps. Specific steps to be investigated include, the kinetics of nucleotide incorporation with questions related to defining the rate limiting steps in correct nucleotide incorporation, the definition of the role of transcript position, sequence, and length on the kinetics of correct and incorrect nucleotide incorporation, the linkage of these kinetic steps to the manifestation of overall transcription fidelity, the rate-limiting steps in nucleotide excision by the polymerase, how the enzyme complex partitions between transcript cleavage and pyrophosphorolysis during nucleotide excision, and again the effect of transcript position, sequence, and length on the kinetics of nucleotide excision.