Enzymology of a Catalytic RNA Molecule

这个项目的长期目标是从根本上了解四膜虫 核酶实现其巨大的速率提高，以及这个内含子如何执行一系列复杂的步骤， 这是精确和有效连接外显子所必需的。希望这种深入了解将进一步促进 对生物催化和RNA有了更全面的了解。此外，核酶在 研究作为体内特异性RNA靶向破坏的潜在治疗剂， 这项工作提供的基本见解将有助于设计这种治疗性RNA。具体目标 在未来五年内，我们的目标如下： 1.四膜虫核酶反应的动力学和热力学描述将进一步发展， 探索RNA催化的具体机制问题，并解决这个内含子如何发挥作用， 多步自剪接反应。对单个反应步骤的这种详细分析对于解剖 催化策略和理解复杂分子过程中的功能，如自我剪接。这 自剪接反应可能为RNA参与更复杂的过程提供了一个模型，例如前- mRNA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 被覆盖在RNA的带电磷酸二酯骨架上的金属离子“海洋”所掩盖。最近的研究 确定了一套新的金属离子/基板相互作用，涉及三个活性位点的金属离子。的身份 现在将探测与这些活性位点金属离子配位的核酶上的官能团，金属离子也将被探测 离子/底物相互作用在各个反应步骤。 3.过渡态类似物在理解蛋白酶的相互作用方面是有价值的， 负责过渡态稳定。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;在确定RNA的能力， 通过分子内的催化作用;并提供了一个稳定的过渡态相互作用模型， 核酶活性构象的结构表征。双底物的结合特性 用3 ′，3 ′-磷酸二酯键合成的类似物和核酶的钒基过渡态类似物 将被确定。 3业绩地点（组织、城市、州） 斯坦福大学 斯坦福大学 关键人员。参见第11页的说明。根据需要使用续页，以下列格式提供所需信息。 名称组织在项目中的角色 丹尼尔赫施拉格斯坦福大学首席研究员 朱棣文斯坦福大学联合研究员 R.加州大学戴维斯分校大卫布里特 Joseph Piccirilli芝加哥大学联合研究员 PHS 398（Rev. 4/98）第2 BB页 在整个应用程序的底部连续编号页面。DoQL使用后缀，如3a，35。 抄送校长/项目主任（最后一位，第一位，中间一位）：Herscl： 在每个打印页和Snuation页的顶部键入主要研究者/项目负责人的姓名。(For型号规格，见 第6页的说明）。 研究资助 目录 页码 首页1 说明，

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,