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,

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Jones, Warren
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Gleitsman, Kristin R; Sengupta, Raghuvir N; Herschlag, Daniel (2017) Slow molecular recognition by RNA. RNA 23:1745-1753
Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem et al. (2017) Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution. J Biol Chem 292:20960-20974
Sengupta, Raghuvir N; Van Schie, Sabine N S; Giamba?u, George et al. (2016) An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions. RNA 22:32-48
Chen, Bob; Lim, Sungwon; Kannan, Arvind et al. (2016) High-throughput analysis and protein engineering using microcapillary arrays. Nat Chem Biol 12:76-81
Sunden, Fanny; AlSadhan, Ishraq; Lyubimov, Artem Y et al. (2016) Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily. J Am Chem Soc 138:14273-14287
van Schie, Sabine N S; Sengupta, Raghuvir N; Herschlag, Daniel (2016) Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes. PLoS One 11:e0160457
Shi, Xuesong; Bisaria, Namita; Benz-Moy, Tara L et al. (2014) Roles of long-range tertiary interactions in limiting dynamics of the Tetrahymena group I ribozyme. J Am Chem Soc 136:6643-8
Gleitsman, Kristin R; Herschlag, Daniel H (2014) A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction. RNA 20:1732-46
Shi, Xuesong; Herschlag, Daniel; Harbury, Pehr A B (2013) Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations. Proc Natl Acad Sci U S A 110:E1444-51
Frederiksen, John K; Li, Nan-Sheng; Das, Rhiju et al. (2012) Metal-ion rescue revisited: biochemical detection of site-bound metal ions important for RNA folding. RNA 18:1123-41

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