Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.