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代谢中起重要作用， 包括RNA剪接、翻译和基因调控的核心功能 表情尽管这些蛋白质很重要，但它们的RNA解旋酶 活性尚未进行酶学研究。基础知识 因此，我们对细胞代谢的了解有限， RNA解旋酶家族中马达蛋白的反应机制。解决 针对这一问题，已经对两种病毒DexH/D启动了机制研究。 蛋白质：来自牛痘的NPH-II和来自丙型肝炎病毒（HCV）的NS 3 -4A。的 NPH-Ⅱ蛋白是一种进行性定向RNA解旋酶， ATP结合和水解的特定作用。建立了 NPH-II活动的质量特征，本提案旨在直接使用 停止流动动力学测量以确定定量动力学 参数如易位速率，反应步长，持续合成能力， 解旋酶结合，ATP结合和水解速率常数，描述了 这种原型RNA解旋酶的催化活性的框架。在 此外，RNA和解旋酶之间分子识别的决定因素 将建立。为了确定这些发现是否具有普遍性， 解旋酶的研究是针对一种对公众构成严重威胁的病毒系统 为了确保健康，将为HCV开发一个补充机制框架 NS 3 -4A蛋白。这种解旋酶也将成为生物物理分析的对象 建立ATP水解循环和易位步骤之间的联系 解旋酶蛋白质。机械信息将促进有意义的 HCV抑制剂和抗病毒治疗的研究， 晶体结构将为结构/功能工作奠定基础， NS 3 -4A蛋白的突变体。NS 3 -4A蛋白也很有用，因为它是 解旋酶功能的新机制研究的一个有前途的候选人， 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.