Kinetic Characterization of the Second Step of Group II Intron Splicing:  Role of Metal Ions and the Cleavage Site 2‘-OH in Catalysis

• Published in: Biochemistry (Easton) Vol. 39; no. 42; pp. 12939 - 12952
• Main Authors: Gordon, Peter M, Sontheimer, Erik J, Piccirilli, Joseph A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 24.10.2000
• Subjects: Binding Sites; Catalysis; Electron Transport Complex IV - genetics; Exons; Hydrogen Bonding; Hydrolysis; Introns; Kinetics; Manganese - chemistry; Metals - chemistry; Nucleic Acid Conformation; RNA Splicing; RNA, Catalytic - chemistry; RNA, Catalytic - genetics; sulfur; Sulfur - chemistry; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001089o

The ai5γ group II intron from yeast excises itself from precursor transcripts in the absence of proteins. When a shortened form of the intron containing all but the 3‘-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3‘ splice site oligonucleotide, a nucleotidyl transfer reaction occurs that mimics the second step of splicing. As this tripartite 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 previously upon sulfur substitution of the 3‘-oxygen leaving group and to elucidate by atomic mutagenesis the role of the neighboring 2‘-OH in catalysis. The results suggest that both the 3‘-oxygen leaving group and the neighboring 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 specificity 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, raising the possibility that a single metal ion mediates interactions with the 2‘- and 3‘-oxygen atoms at the 3‘ splice site.