The group I self-splicing introns, exemplified by the Tetrahymena ribosomal RNA intron, are RNA enzymes (ribozymes) that catalyze phosphodiester exchange reactions. This proposal is focused on the analysis of substrate binding by this class of RNA enzymes. Previous experiments have led to the identification of part of the guanosine binding site of this RNA enzyme. Here, methods for further defining the guanosine binding site are presented, and novel approaches to defining the RNA substrate binding site are described. These experiments use a recently described system for the in vitro genetic analysis of interactions within the ribozyme. Several enhancements of these in vitro selection and amplification methods are proposed, along with plans to use these techniques extensively in the study of ribozyme-substrate interactions. Potential enzymesubstrate interactions that are identified will be studied by the kinetic analysis of mutant enzymes in combination with chemically synthesized substrate analogue.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045315-02
Application #
3304744
Study Section
Biochemistry Study Section (BIO)
Project Start
1991-07-01
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199