The effect of diverse metal ions on hatchet ribozyme activity. (A) Single time-point assays evaluating the ability of various divalent metal ions to support Ht-2 ribozyme activity. Reactions were incubated with 1 mM of the divalent metal ion as indicated for 30 min. Other experimental details and annotations are as described in the legend to Figure 1C. (B) Ht-2 ribozyme activity with certain divalent metal ions at 0.5 mM in the absence or presence of Mg2+. Other details are as described in Figure 1A. (C) The effect of cobalt hexammine on the function of Ht-2. Reactions were incubated in the absence (−) or presence (+) of 5 mM [Co(NH3)6]3+ and/or 5 mM Mg2+ as indicated. Other experimental details and annotations are as described in the legend to Figure 1C. (D) Sites of sulfur substitution (shaded) to generate the Rp or Sp isoforms of the ribozyme substrate. (E) Mass spectrometry data including the calculated (calc.) mass of the phosphorothioate substrate and the observed (obs.) mass of the sample. (F) Plot of the natural logarithm of the fraction of substrate RNA remaining uncleaved versus incubation time. O and S designate unmodified and phosphorothioate substrate RNAs, respectively. Of note, 10 mM MgCl2 or 10 mM MnCl2 were present in the reaction mixtures. Other conditions were as described in the legend to Figure 1C.
