Identification of an active site ligand for a group I ribozyme catalytic metal ion.
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 (R(P) or S(P)) 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 S(P), C208 S(P), A306 R(P), and A306 S(P)) exhibited a significant reduction in the cleavage rate. Only the effect of the C208 S(P) phosphorothioate substitution could be significantly rescued by the addition of a thiophilic metal ion, either Mn(2+) or Zn(2+), when tested with an all-oxy substrate. The effect was not rescued with Cd(2+). To determine if one of the catalytic metal ions is coordinated to the C208 pro-S(P) oxygen, the phosphorothioate-substituted ribozymes were also assayed using oligonucleotide substrates with a 3'-phosphorothiolate or an S(P) phosphorothioate substitution at the scissile phosphate. This resulted in a second metal specificity switch, in that Mn(2+) or Zn(2+) no longer rescued the C208 S(P) ribozyme, but Cd(2+) provided efficient rescue in the context of either sulfur-containing substrate. The 3'-oxygen and the pro-S(P) oxygen of the scissile phosphate are both known to coordinate the same metal ion, M(A), which stabilizes the negative charge on the leaving group 3'-oxygen in the transition state. Taken together, these data suggest that metal M(A) is coordinated to the C208 pro-S(P) 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.
Szewczak
Alexander A
AA
Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06520-8114, USA.
Kosek
Anne B
AB
Piccirilli
Joseph A
JA
Strobel
Scott A
SA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D008024:Ligands; D008670:Metals / Q000378:metabolism*; D011487:Protein Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D013768:Tetrahymena / Q000201:enzymology / Q000235:genetics; D013873:Thionucleotides / Q000378:metabolism
D017931:DNA Primers; D008024:Ligands; D008670:Metals; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D013873:Thionucleotides