Structure and Catalysis of an RNA Enzyme

RNA 酶的结构和催化

• 批准号: 6470415
• 负责人: William G Scott
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6470415
• 关键词: RNA; X ray crystallography; active sites; catalyst; chemical cleavage; conformation; crystallization; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; high performance liquid chromatography; nuclear magnetic resonance spectroscopy; protein structure function; ribozymes

DESCRIPTION (provided by applicant): The global objective of the research proposed here is to understand the fundamental question of how a small RNA enzyme, called the hammerhead ribozyme, works. Using static X-ray crystallography, dynamic crystallographic intermediate trapping experiments (a form of time-resolved crystallography), together with a solid-state NMR collaboration and a variety of biochemical approaches, the hypothesis that that the RNA molecule itself, rather than simply acting as a relatively passive scaffold for binding metal ions, actively participates in the chemistry of catalysis, will be tested in a variety of ways. In doing so, an understanding of the relationship between catalytic RNA structure and function (i.e., catalysis) will be obtained. The specific aims of the program described in this research proposal are: (1) to elucidate the forces that drive and stabilize the conformational change known to be required for catalysis in the hammerhead ribozyme structure; (2) to deduce the structure of the hammerhead-ribozyme transition-state; (3) to characterize definitively the catalytic activity of the crystallized hammerhead ribozyme to better understand the mechanistic implications of the structural changes previously observed in our time-resolved crystallographic experiments; and (4) to test our understanding of ribozyme catalysis by designing new catalytic RNAs. Each of these four specific aims is designed to probe, independently, the cleavage mechanism of the hammerhead ribozyme from a variety of viewpoints. The potential use of hammerhead ribozymes as therapeutic agents that target RNA viruses (such as HIV) and pathological mRNAs (such as oncogene transcripts) is well-documented. Although our primary motive for the research proposed here is to answer questions of a fundamental scientific nature, it is hoped that the results of these studies will provide practical information to the scientific and medical communities to enable more potent and effective ribozyme-based pharmaceuticals to be developed by others.

描述（由申请人提供）：研究的全球目标 这里提出的是理解一个基本问题， 一种叫做锤头状核酶的酶起作用。使用静态X射线 晶体学，动态晶体学中间捕获实验（a 时间分辨晶体学的形式），连同固态NMR 合作和各种生化方法，假设， RNA分子本身，而不是简单地充当相对被动的 结合金属离子的支架，积极参与化学 催化剂，将以各种方式进行测试。在这样做的时候， 催化RNA结构和功能之间的关系（即， 催化剂）将获得。本报告所述方案的具体目标 研究建议是：（1）阐明驱动和稳定 已知锤头状结构中催化所需的构象变化 （2）推导锤头状核酶的结构 （3）明确表征催化剂的催化活性 结晶锤头状核酶，以更好地了解 以前在我们的时间分辨中观察到的结构变化的影响 晶体学实验;（4）测试我们对核酶的理解 通过设计新的催化RNA来催化。这四个具体目标中的每一个都是 设计成独立探测锤头鲨的分裂机制 从不同的角度来看。锤头鲨的潜在用途 核酶作为靶向RNA病毒（如HIV）的治疗剂， 病理性mRNA（如癌基因转录物）是有据可查的。虽然 我们在这里提出的研究的主要动机是回答一个 基本的科学性质，希望这些研究的结果 将为科学和医学界提供实用信息， 使得能够开发更有效的基于核酶的药物 被人