Enzymology of a Catalytic RNA Molecule

这个项目的长期目标是从根本上理解四膜虫是如何 核酶实现了其巨大的速率提高，以及该内含子如何执行一系列复杂的步骤 准确有效地连接外显子所必需的。希望这种深入的了解将进一步 对生物催化和核糖核酸有更全面的了解。此外，核酶还在 研究作为在体内靶向破坏特定RNA的潜在治疗方法，并且有可能 这项工作提供的基本见解将有助于此类治疗性RNA的设计。具体目标 未来五年的情况如下： 1.四膜虫核酶反应的动力学和热力学描述将进一步发展到 探索RNA催化的具体机制问题，并解决该内含子如何发挥作用以实现 多步自剪接反应。这种对单个反应步骤的详细分析对解剖至关重要 催化策略和理解复杂分子过程中的功能，如自剪接。这 自剪接反应可能为RNA参与更复杂的过程提供一种模型，如前 信使核糖核酸剪接和翻译。 2.二价金属离子对RNA折叠和功能至关重要，但单个金属的作用通常是 被包裹在带电的RNA磷酸二酯主干上的金属离子的“海”遮挡住了。最近的研究表明 确定了一组新的涉及三个活性中心金属离子的金属离子/底物相互作用。的身份 现在将探测核酶上与这些活性中心金属离子配位的官能团，金属也是如此 个别反应步骤中的离子/底物相互作用。 3.过渡态类似物对于理解蛋白质酶之间的相互作用是有价值的 负责过渡状态的稳定。模拟核酶过渡态的类似物 反应可能有助于理解催化的能量学；有助于确定RNA提供 通过分子内催化；并提供过渡态相互作用的稳定模型，这将允许 核酶活性构象的结构表征。双取代基团的结合性能 用3‘，3’-磷酸二酯键合成的类似物，以及核酶的钒过渡态类似物 将会被确定。 3格式站点(S)(组织、市、州) 斯坦福大学 加利福尼亚州斯坦福 关键人员。请参阅第11页的说明。根据需要使用第二页以如下所示的格式提供所需信息。 命名项目中的组织角色 丹尼尔·赫施拉格斯坦福大学首席研究员 朱斯坦福大学联合研究员 戴维斯，加州大学R.David Britt大学共同研究员 约瑟夫·皮奇里利，芝加哥大学联合研究员 小灵通398(4/98版)第2页BB 在整个应用程序的底部连续编号页码。DoQQL使用后缀，如3a，35。 CC Princj调查员/项目总监(最后、第一、中间)：Herscl： 在每个打印页面和评估页面的顶部键入首席调查员/项目主管的姓名。(有关类型规格，请参阅 第6页上的说明。) 研究补助金 目录 页码 正面第1页 描述，

The long term goal of this project is to understand, on a fundamental level, how the Tetrahymena ribozyme achieves its enormous rate enhancement and how this intron carries out the complex series of steps required for the accurate and efficient ligation of exons. It is hoped that such in-depth understanding will further more general understanding of both biological catalysis and RNA. In addition, ribozymes are under investigations as potential therapeutics for the targeted destruction of specific RNAs in vivo, and it is possible that fundamental insights provided by this work will aid in the design of such therapeutic RNAs. Specific aims for the next five years are as follows: 1. The kinetic and thermodynamic description of Tetrahymena ribozyme reactions will be further developed to probe specific mechanistic questions of RNA catalysis and to address how this intron functions to carry out the multi-step self-splicing reaction. Such detailed analysis of individual reaction steps is crucial for dissecting catalytic strategies and for understanding function in a complex molecular process such as self-splicing. This self-splicing reaction may provide a model for the involvement of RNA in more complex processes such as pre- mRNA splicing and translation. 2. Divalent metal ions are crucial to RNA folding and function, but the role of individual metal is typically obscured by the 'sea' of metal ions that coat the charged phosphodiester backbone of RNA. Recent studies have identified a novel set of metal ion/ substrate interactions involving three active site metal ions. The identity of functional groups on the ribozyme that coordinate these active site metal ion will now be probed, as will metal ion/ substrate interactions in individualreaction steps. 3. Transition state analogs have been valuable in understanding interactions of protein enzymes that are responsible for transition state stabilization. Analogs that mimic aspects of the transition state for the ribozyme reaction may help in understanding the energetics of catalysis; in determining the ability of RNA to provide catalysis via intramolecularity; and in providing a stable model of transition state interactions that will allow structural characterization of the active conformation of the ribozyme. The binding properties of bisubstrate analogs, synthesized with 3',3'-phosphodiester linkages, and vanadyl transition state analogs for the ribozyme will be determined. 3ERFORMANCE SITE(S) (organization, city, state) Stanford University Stanford, CA KEY PERSONNEL. See instructions on Page 11. Usecontinuationpages as neededto provide the required information in the format shown below. Name Organization Role on Project Daniel Herschlag Stanford University Principal Investigator Steven Chu Stanford University Co-Investigator R. David Britt Universityof California, Davis Co-Investigator Joseph Piccirilli The Universityof Chicago Co-Investigator PHS 398 (Rev. 4/98) Page 2 BB Number pages consecutively at the bottom throughout the application. DoQQLuse suffixes such as 3a, 35. cc Princij 'estigator/Program Director (Last, first, middle): Herscl: Type the name of the principal investigator/program director at the top of each printed page and Snuation page. (For type specifications, see instructions on page 6.) RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,