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.