Although best known for self-splicing ability, groups I introns can also catalyze other reactions such as ligation of oligonucleotides, circularization of excised intron RNA, and hydrolysis at the 3' splice site. The Didymium rDNA intron DiSSU1 contains two distinct ribozymes, GIR1 and GIR2. GIR1 is a novel group I intron in that it is the first ribozyme whose putative catalytic function is hydrolysis, and is the smallest (approximately 150 nt) identified to date. Three different but complementary methods, kinetics, in vitro selection, and x-ray crystallography, will be used to characterize GIR1. Conditions for optimal catalysis and the kinetics of hydrolysis will be determined. In vitro selection methods will be used to select for GIR1-like ribozymes with increased catalytic efficiency. In addition, in order to further understand the minimal structural features required for self-splicing, GIR1 will be converted into a self-splicing ribozyme, and subjected to in vitro selection. These methods will provide sequence data for comparative sequence analysis and modeling, and provide a better method for obtaining homogeneous pools of RNA for use in crystallization trials. Lastly, crystallization of Didymium GIR1, in vitro selected GIR1 ribozymes, as well as Naegleria GIR1 will be initiated. Increased understanding of the catalytic mechanism and die structural requirements for hydrolysis by GIR1 will facilitate the design of group I ribozymes with altered or improved activity for therapeutic use.
