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Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4–P6 domain

  • SOUMITRA BASU (a1) and SCOTT A. STROBEL (a1)

Abstract

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 α-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+ (≥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.

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Corresponding author

Reprint requests to: Scott A. Strobel, Department of Molecular Biophysics and Biochemistry, Department of Chemistry, Yale University, PO Box 208114, 260 Whitney Avenue, New Haven, Connecticut 06520-8114, USA; e-mail: strobel@csb.yale.edu.

Keywords

Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4–P6 domain

  • SOUMITRA BASU (a1) and SCOTT A. STROBEL (a1)

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