Kinetic characterization of the second step of group II intron splicing: Role of metal ions and the cleavage site 2 '-OH in catalysis

The ai5 gamma group II intron from yeast excises itself from precursor tran scripts in the absence of proteins. When a shortened form of the intron con taining all but the 3'-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3' splice site oligonucleotide, a nucleotidyl transf er reaction occurs that mimics the second step of splicing. As this tripart ite reaction provides a means to identify important functional groups in 3' splice site recognition and catalysis, we establish here a minimal kinetic framework and demonstrate that the chemical step is rate-limiting. We use this framework to characterize the metal ion specificity switch observed pr eviously upon sulfur substitution of the 3'-oxygen leaving group and to elu cidate by atomic mutagenesis the role of the neighboring 2'-OH in catalysis . The results suggest that both the 3'-oxygen leaving group and the neighbo ring 2'-OH are important ligands for metal ions in the transition state but not in the ground state and that the 2'-OH may play an additional role in transition state stabilization by donating a hydrogen bond. Metal specifici ty switch experiments combined with quantitative analysis show that the Mn2 + that interacts with the leaving group binds to the ribozyme with the same affinity as the metal ion that interacts with the neighboring 2'-OH, raisi ng the possibility that a single metal ion mediates interactions with the 2 '- and 3'-oxygen atoms at the 3' splice site.