2'-Hydroxyl groups important for exon polymerization and reverse exon ligation reactions catalyzed by a group I...

The functional importance of ribose moieties in both exons and in intron sequences proximal to the 3' splice site of a group I intron has been analyzed using a novel exon polymerization reaction. The ribozyme is a modified version of a self-splicing bacterial tRNA intron (I) that attacks a 20-nucleotide synthetic ligated exon substrate (E1.E2), yielding E1 and I.E2 by reverse exon ligation. A series of repetitive reactions then polymerize E2 on the 3' end of the intron; attack by E1 subsequently generates E1.(E2)n. Systematic deoxyribonucleotide substitution within E1.E2 was used to probe the function of 2'-hydroxyl groups in each exon and the 3'-terminal nucleotides of the intron. We find that ribose at the splice junction (U-1) and at the two adjacent positions with E1 (A-2, C-3) is important for reverse exon ligation. Within E2, deletion of 2'-hydroxyl groups of the nucleotides that form P10 does not affect reactivity. In contrast, ribose at the 3' end of the intron is essential for reverse exon ligation, and the presence of a 2'-OH group in each of the nucleotides comprising P9.0[3'] contributes to reaction efficiency. These results support a model in which specific 2'-hydroxyl groups at and adjacent to the reaction sites form tertiary contacts that serve to stabilize interactions with the catalytic core of the ribozyme. Furthermore, they suggest that the mechanism by which guanosine at the 3' end of the intron is activated for reverse exon ligation is the same as that by which guanosine mononucleotide is activated in the first step of splicing.