Structure and Catalysis of an RNA Enzyme

RNA 酶的结构和催化

• 批准号: 6724427
• 负责人: William G Scott
• 金额: $ 29.44万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6724427
• 关键词: RNA; X ray crystallography; catalyst; chemical cleavage; conformation; crystallization; endonuclease; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; phosphoester ligase; 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 answer the question of how the hammerhead ribozyme is able to switch its catalytic activity between that of an RNA endonuclease and that of an RNA ligase; (2) to elucidate the forces that drive and stabilize the conformational change known to be required for catalysis in the hammerhead ribozyme structure; (3) to solve the structure of a newly discovered hammerhead ribozyme construct that possesses stabilizing tertiary structural contacts between helical Stems I and II which increase its catalytic rate 1000-fold compared to previously studied hammerhead RNA sequences, 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）：此处提出的研究的全球目标是了解一个基本问题，即一种小的RNA酶（称为Hammerhead Ribozyme）如何起作用。使用静态X射线晶体学，动态晶体学中间捕获实验（一种时间分辨晶体学的形式），以及固态NMR协作以及多种生化方法，即RNA分子本身，而不是简单地作为一种相对被动地培养的型物质，而不是简单地用作型金属，而不是简单地将其作为一种相关的型号，而不是简单地构成了一定的型号。这样，将了解对催化RNA结构与功能（即催化）之间关系的理解。在本研究建议中描述的程序的具体目的是：（1）回答锤头核酶如何在RNA内核酸酶和RNA连接酶的催化活性之间切换其催化活性的问题； （2）阐明在锤头核酶结构中催化所需的构象变化和稳定构象变化的力； （3）解决了新发现的锤头核酶构建体的结构，该结构具有螺旋茎I和II之间稳定的高等教育结构接触，该结构与先前研究的Hammerhead RNA序列相比增加了其催化速率为1000倍，并且（4）通过设计新的催化催化性催化性催化性RNAS来测试我们对核酶催化的理解。这四个特定目标中的每一个都旨在独立探测锤头核酶的裂解机理，从各种观点。锤头核酶作为治疗剂的潜在用途已充分记录在靶向RNA病毒（例如HIV）和病理mRNA（例如癌基因转录本）的治疗剂。尽管这里提出的研究的主要动机是回答有关基本科学性质的问题，但希望这些研究的结果将为科学和医疗社区提供实用信息，以使其他人开发更多的有效和有效的基于核酶的药物。