Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain.
Divalent metal ions are essential for the folding and catalytic activities of many RNAs. A commonly employed biochemical technique to identify metal-binding sites in RNA is the rescue of Rp alpha-phosphorothioate (PS) interference by the addition of soft divalent metal ions. To access the ability of such experiments to accurately identify metal-ion coordinations within a complex RNA fold, we report metal-rescue results from the Tetrahymena group I intron P4-P6 domain, where the location and coordination of five divalent metal ions have been determined by X-ray crystallography [J.H. Cate et al., Nat Struct Biol, 1997, 4:553]. We used a native gel mobility-shift to assay for P4-P6 folding in the presence of various divalent metal ions, and found that even moderate concentrations of Mn2+ (> or =0.5 mM) can rescue PS interference at sites that do not coordinate metal ions within the P4-P6 crystal structure. To control for such effects, 2'-deoxynucleotide interference was used to titrate the Mn2+ concentration to a level that produces metal-ion-specific rescue (0.3 mM). This concentration of Mn2+ specifically rescued four of the six metal-dependent phosphorothioate effects within the RNA domain, including PS interference resulting from outer-sphere coordination to the metals. Both sites that were not specifically rescued make inner-sphere metal-ion coordinations. Cd2+ and Zn2+ afforded rescue at a smaller subset of the six metal-specific PS sites, though again phosphates making outer-sphere coordinations to metal ions were rescued preferentially. These data on P4-P6 domain folding reinforce the need for caution when interpreting metal-rescue experiments.
Basu
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Strobel
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D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D016054:DNA, Protozoan / Q000235:genetics; D007438:Introns; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016337:RNA, Catalytic / Q000737:chemistry* / Q000187:drug effects*; D016053:RNA, Protozoan / Q000737:chemistry / Q000187:drug effects; D013768:Tetrahymena / Q000235:genetics*; D013873:Thionucleotides / Q000494:pharmacology*
D002413:Cations, Divalent; D016054:DNA, Protozoan; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D013873:Thionucleotides