MECHANISM OF RNA HELICASE ACTIVITY BY DEXH/D PROTEINS

描述(改编自申请人的摘要)：DexH/D的RNA解旋酶 家族在病毒复制和细胞RNA代谢中起着至关重要的作用， 包括在RNA剪接、翻译和基因调控中的中心功能 表情。尽管这些蛋白质很重要，但它们的RNA解旋酶 活性还没有受到酶学研究的影响。基本知识 因此，细胞新陈代谢受到我们对 RNA解旋酶家族中马达蛋白的反应机制。致信地址 针对这一问题，目前已开始对两种病毒DexH/D进行机理研究 蛋白质：来自疫苗的NPH-II和来自丙型肝炎病毒(HCV)的NS3-4A。这个 NPH-II蛋白是一种连续的、定向的RNA解旋酶，具有 结合和水解三磷酸的具体作用。已经确立了 NPH-II活动的质量特征，本提案旨在使用直接和 用于确定定量动力学的停流动力学测量 参数，如移位率、反应步长、加工性、 解旋酶结合、三磷酸腺苷结合和水解速率常数描述 这个典型的RNA解旋酶的催化活性的框架。在……里面 此外，RNA和解旋酶之间分子识别的决定因素 将会建立起来。以确定这些发现是否具有普遍性，并扩展到 解旋酶研究对公众构成严重威胁的病毒系统 卫生，将为丙型肝炎病毒开发一个补充的机制框架 NS3-4A蛋白。这种解旋酶也将是生物物理分析的对象。 建立ATP水解循环和转位步骤之间的联系 解旋酶蛋白。机械性的信息将促进有意义的 丙型肝炎病毒抑制剂和抗病毒治疗的研究，鉴于现有的 晶体结构的研究将为结构/功能研究奠定基础 NS3-4A蛋白的突变体。NS3-4A蛋白也很有用，因为它是 解旋酶功能新机制研究的一个有前途的候选者 在膜结合状态和复杂的大分子机器的背景下。

DESCRIPTION (adapted from applicant's abstract): RNA helicases of the DexH/D family play an essential role in viral replication and cellular RNA metabolism, including central functions in RNA splicing, translation and regulation of gene expression. Despite the importance of these proteins, their RNA helicase activity has not been subjected to enzymological study. Basic knowledge of cellular metabolism is therefore constrained by our limited understanding of reaction mechanism by motor proteins in the RNA helicase family. To address this problem, mechanistic studies have been initiated on two viral DexH/D proteins: NPH-II from Vaccinia and NS3-4A from Hepatitis C Virus (HCV). The NPH-II protein is show to be a processive, directional RNA helicase with specific roles for both the binding and hydrolysis of ATP. Having established qualitative features of NPH-II activity, this proposal aims to use direct and stopped flow kinetic measurements to determine the quantitative kinetic parameters such as translocation rates, reaction step size, processivity, helicase binding, ATP binding and hydrolytic rate constants that describe the framework for catalytic activity of this prototypical RNA helicase. In addition, the determinants for molecular recognition between RNA and helicase will be established. To determine if these findings are general and to extend the helicase studies to a viral system that poses a grave threat to public health, a complementary mechanistic framework will be developed for the HCV protein NS3-4A. This helicase will also be the subject of biophysical analyses to establish the link between cycles of ATP hydrolysis and translocative steps of the helicase protein. The mechanistic information will facilitate meaningful studies on HCV inhibitors and antiviral therapies and, given the availability of a crystal structure will set the stage for structure/function work on mutants of the NS3-4A protein. The NS3-4A protein is also useful because it is a promising candidate for novel mechanistic studies on helicase function in membrane-bound states and in the context of complex macromolecular machines.