MECHANISM OF RNA HELICASE ACTIVITY BY DEXH/D PROTEINS

DEXH/D 蛋白激活 RNA 解旋酶的机制

• 批准号: 6644858
• 负责人: Anna Marie Pyle
• 金额: $ 21.98万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-12-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6644858
• 关键词: chemical kinetics; enzyme activity; enzyme mechanism; helicase; hepatitis C virus; vaccinia virus; virus RNA; virus protein; virus replication

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.

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代谢中发挥重要作用， 包括 RNA 剪接、翻译和基因调控的核心功能 表达。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，细胞代谢受到我们有限的了解的限制 RNA解旋酶家族中运动蛋白的反应机制。致地址 针对这个问题，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 NS3-4A。这 NPH-II 蛋白被证明是一种持续的、定向的 RNA 解旋酶，具有 ATP 的结合和水解的特定作用。已建立 NPH-II 活动的定性特征，该提案旨在使用直接和 停流动力学测量以确定定量动力学 参数，例如易位速率、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 该原型 RNA 解旋酶催化活性的框架。在 此外，RNA 和解旋酶之间分子识别的决定因素 将成立。确定这些发现是否具有普遍性并扩展 对对公众构成严重威胁的病毒系统进行解旋酶研究 健康方面，将为 HCV 开发一个补充机制框架 NS3-4A 蛋白。该解旋酶也将成为生物物理分析的主题 建立 ATP 水解循环和易位步骤之间的联系 解旋酶蛋白。机械信息将有助于有意义的 HCV 抑制剂和抗病毒疗法的研究，并且考虑到可用性 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 解旋酶功能新机制研究的有希望的候选者 膜结合态和复杂大分子机器的背景。