Identification of an active site ligand for a group I ribozyme catalytic metal ion

Abstract

The transition state of the group I intron self-splicing reaction is stabilized by three metal ions. The functional groups within the intron substrates (guanosine and an oligoribonucleotide mimic of the 5′-exon) that coordinate these metal ions have been systematically defined through a series of metal ion specificity switch experiments. In contrast, the catalytic metal ligands within the ribozyme active site are unknown. In an effort to identify them, stereospecific (RP or SP) single-site phosphorothioate substitutions were introduced at five phosphates predicted to be in the vicinity of the catalytic center (A207, C208, A304, U305, and A306) within the Tetrahymena intron. Of the 10 ribozymes that were studied, four phosphorothioate substitutions (A207 SP, C208 SP, A306 RP, and A306 SP) exhibited a significant reduction in the cleavage rate. Only the effect of the C208 SP phosphorothioate substitution could be significantly rescued by the addition of a thiophilic metal ion, either Mn2+ or Zn2+, when tested with an all-oxy substrate. The effect was not rescued with Cd2+. To determine if one of the catalytic metal ions is coordinated to the C208 pro-SP oxygen, the phosphorothioate-substituted ribozymes were also assayed using oligonucleotide substrates with a 3′-phosphorothiolate or an SP phosphorothioate substitution at the scissile phosphate. This resulted in a second metal specificity switch, in that Mn2+ or Zn2+ no longer rescued the C208 SP ribozyme, but Cd2+ provided efficient rescue in the context of either sulfur-containing substrate. The 3′-oxygen and the pro-SP oxygen of the scissile phosphate are both known to coordinate the same metal ion, MA, which stabilizes the negative charge on the leaving group 3′-oxygen in the transition state. Taken together, these data suggest that metal MA is coordinated to the C208 pro-SP phosphate oxygen, which constitutes the first functional link between a specific catalytic metal ion and a particular functional group within the group I ribozyme active site.