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: 6387074
• 负责人: Anna Marie Pyle
• 金额: $ 22.96万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 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 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387074
• 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 家庭在病毒复制和细胞RNA代谢中起着至关重要的作用， 包括RNA剪接，基因翻译和调节中的中心功能 表达。尽管这些蛋白质很重要，但其RNA解旋酶 活性尚未接受酶学研究。基本知识 因此，细胞代谢受到我们对 RNA解旋酶家族中运动蛋白的反应机理。解决 这个问题，机械研究已经在两个病毒DEXH/D上启动 蛋白质：疫苗的NPH-II和丙型肝炎病毒（HCV）的NS3-4A。这 NPH-II蛋白被证明是一种定向RNA解旋酶， ATP结合和水解的特定作用。建立了 NPH-II活动的定性特征，该建议旨在使用直接和 停止流动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，加工性， 解旋酶结合，ATP结合和水解速率常数描述 该原型RNA解旋酶的催化活性框架。在 补充，RNA和解旋酶之间的分子识别决定因素 将建立。确定这些发现是否一般并扩展 对病毒系统的解旋酶研究对公众构成严重威胁 健康，将为HCV开发一个互补的机械框架 蛋白质NS3-4A。该解旋酶也将是生物物理分析的主题 建立ATP水解循环与易位步骤之间的联系 解旋酶蛋白。机械信息将促进有意义的 有关HCV抑制剂和抗病毒疗法的研究，并鉴于可用性 晶体结构将为结构/功能的工作奠定基础 NS3-4A蛋白的突变体。 NS3-4A蛋白也很有用，因为它是 一个有前途的有关解旋酶功能的新机械研究的有前途的候选者 膜结合状态和复杂的大分子机器的背景。