Metal ion dependency of a pistol ribozyme. (A, left) Pistol ribozyme cleavage assays with the L. sphaericus bimolecular construct (Fig. 3A) incubated for 30 min in absence (−) or presence (+) of various divalent metal ions at concentrations of 1 mM. (Right) Incubation of the bimolecular pistol construct for 30 min with 0.5 mM divalent metal ions alone or in combination with equal amounts of MgCl2. (B) Incubation of the same bimolecular construct in the absence (none) or presence of different monovalent cations at 1 M for 1 h. To chelate contaminating divalent metal ions, 30 mM EDTA was added to these reactions. (C) Reactions of the bimolecular complex in the absence (−) or presence (+) of 5 mM cobalt hexammine chloride [Co(NH3)6Cl3] or MgCl2 for 1 h in the presence of 5 mM EDTA. (D) Chemical structure of a phosphorothioate RNA linkage. The gray circles highlight the fact that the phosphorothioate substrate analogs exist as a mixture of two isomers, RP and SP. (E) Mass spectrum depicting the A. putredinis phosphorothioate substrate RNA for the bimolecular pistol ribozyme construct. The correlation between the calculated (calc.) and observed (obs.) masses of the modified RNA is consistent with the presence of a sulfur atom. (F) Self-cleavage analysis of the L. sphaericus pistol ribozyme with either a phosphate (O) or phosphorothioate (S) linkage at the cleavage site depicted by a plot of the natural logarithm of the fraction of substrate remaining versus time. Cleavage assays were performed in standard reactions conditions with either 1 mM MgCl2 or 0.1 mM MnCl2. Dashed lines designate y-axis values that represent 50% or 90% cleavage of the substrate.
