The main long term goals of this project are to understand, in a fundamental sense, the catalytic strategies of RNA enzymes, and to understand the physical and chemical principles underlying this catalysis. In addition, catalysis provides a sensitive probe of structure-function relationships of RNA, some of which may be general to other RNA molecules. Finally, this understanding may aid in the design of RNA enzymes that can specifically and efficiently cleave RNA targets in vivo. The RNA enzyme or """"""""ribozyme"""""""" derived from the intervening sequence of Tetrahymena pre-rRNA, which catalyzes a site-specific endonuclease reaction analogous to the first step of self-splicing, has been chosen for study because it is the best characterized RNA catalyst. Thus, further investigation provides the opportunity to unravel fundamental aspects of RNA catalysis.
Specific aims are as follows: 1. Directly determine the rate constant of the chemical step of the Tetrahymena ribozyme reaction using rapid quench techniques. Investigate the origin of an apparent pKa near 7. Perform additional measurements to complete the kinetic and thermodynamic framework of the ribozyme reaction. This allows isolation of individual reaction steps, which is crucial for dissecting the catalytic strategies of the ribozyme. 2. Vary in a systematical and rational fashion, the reaction substrates (the guanosine nucleophile and the oligonucleotide that is cleaved) in order to reveal the types of interactions important in binding and catalysis. Analysis of these series of substrates with mutant ribozymes (generated both by site-directed mutagenesis and in vitro selection) will reveal further aspects of these interactions and their energetics. 3. Compare the binding of bisubstrate and transition state analogs to the binding of the individual substrates and products. These comparisons and the results from the experiments of Specific Aim 2 are expected to provide insight into the catalytic strategies employed by the ribozyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM049243-01
Application #
3308591
Study Section
Biochemistry Study Section (BIO)
Project Start
1993-05-01
Project End
1998-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
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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
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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
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; Peck, Ariana; Salzman, Julia et al. (2015) Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site. Elife 4:
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; 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

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