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,