9458185 Pyle This research program is designed to elucidate the tertiary folding patterns and chemical mechanism of the group II intron, a structurally complex catalytic RNA involved in eukaryotic gene expression. Group II introns are often required for the processing of pre-RNAs in the mitochondria of plants and yeast. The tertiary architecture, or folding of the group II intron is of great interest because it cannot be solved using conventional base-pairing rules and preliminary work suggests it is different from any RNA studied previously. The specific objectives being studied are: 1) to determine the location an identity of tertiary interactions stabilizing group II intron folding. In particular, characterize interactions between Domain (D1), which binds the 5'-exon, and Domain 5 (D5), a small catalytically essential RNA that binds D1 in-trans. 2) Provide a complete kinetic framework for the folding pathway and chemical mechanism of the wild-type group II intron, enabling a precise interpretation of mutational results. 3) Solve the three-dimensional structure of the group II intron through a combination of biochemical, X-ray crystallographic and molecular modeling studies. 4) Utilize Group II intron substructures derived from catalytic D5 and nucleophilic D6 as covalent probes to scan for group II introns in humans. 5) By swapping small nuclear RNA components Or the spliceosome with domains of the Group II intron, search for evidence that group II introns and spliceosomal RNAs are related in structure or function. %%% This research program is investigating the folding patterns and chemical mechanisms of the Group II intron, which is a structurally complex catalytic RNA involved in gene expression in plants and yeast. The folding of the group II intron is of great interest because it cannot be solved using conventional base-pairing rules for nucleic acids and preliminary work suggests it is different from any RNA studied previously. These studies may serve a s an interesting and biologically relevant model for novel forms of RNA tertiary organization that are stabilized through nucleic acid base-backbone contacts, metal ion bridges, and other unusual inter actions. Catalytic RNAs (ribozymes) such as the group II intron provide a particularly effective means of characterizing RNA tertiary interactions because ribozymes report on changes in their structure through changes in their catalytic activity. These studies will provide kinetic evidence for group II intron folding as well as solve the three-dimensional structure of the group II intron. Studies will also be done to scan for human group II introns to see if they occur in animal cells which is not known to date. ***
