MUTAGENESIS AND COMPARATIVE SEQUENCE-ANALYSIS OF A BASE TRIPLE JOINING THE 2 DOMAINS OF GROUP-I RIBOZYMES

Tertiary interactions are important in the higher-order folding of cat alytic RNAs. Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanob acterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila. T his base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7. We made base pair and single-nucle otide substitutions in the fifth base pair of P4, a G-C in the wild-ty pe Anabaena intron, and tested them for self-splicing activity. The re sults suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7. Comparative sequ ence analysis was used to determine the different combinations of base triples that occur in similar to 450 natural group I introns identifi ed to date. About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model. Disrupting this base triple in t he Tetrahymena intron resulted in the disappearance of splicing interm ediates (intron 3' exon and 5' exon), even though the first step of sp licing was not affected. Restoration of the base triple by a compensat ory mutation reverted the intermediates to wild-type levels. These res ults suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding th e second step. Repositioning of the base triple to form a new set of i nteractions may be required for the second step of splicing.