Kinetic characterization of the second step of group II intron splicing: role of metal ions and the cleavage site 2'-OH in catalysis.
The ai5gamma 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 Mn(2+) 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.
Gordon
P M
PM
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, Illinois 60637, USA.
Sontheimer
E J
EJ
Piccirilli
J A
JA
D001665:Binding Sites; D002384:Catalysis; D003576:Electron Transport Complex IV / Q000235:genetics; D005091:Exons; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007438:Introns; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics*; D013455:Sulfur / Q000737:chemistry; D013816:Thermodynamics
D008670:Metals; D016337:RNA, Catalytic; D008345:Manganese; D013455:Sulfur; D003576:Electron Transport Complex IV