Assays of the ribozymes in various divalent ion concentrations. (A) Preliminary pick-up-the-tail assays were performed with 2.5-fold substrate excess as described in the text for activity after 60 minutes at 37°C. (Note: the original selection of the PV ribozyme  was performed at 3.25 h, at which time the PV activity surpasses that of the CG activity). The fraction of the initial amount of ribozyme RNA that reacted under conditions of 2.5-fold excess substrate was measured for each variant in the absence (green lines) or presence (red lines) of 10 mM CaCl2 under concentrations of MgCl2 ranging from 0.5–25 mM. Above 5 mM no increase in activity was observed. In the absence of a CaCl2 a slight inhibition of activity with increasing MgCl2 was observed above 0.1 mM, as noted before . From these data, the conditions for formal analysis of interpretive behaviour were chosen near the endpoints of these curves: at, and well above, the point of maximum separation (0.5 mM MgCl2, indicated with the dashed black line). These conditions were 25 mM MgCl2 + 10 mM CaCl2 (O1), 0.5 mM MgCl2 + 10 mM CaCl2 (O2), 25 mM MgCl2 (O3), and 0.5 mM MgCl2 (O4). (B) Example gels of ribozyme reacted for 0–60 min with 2.5-fold excess S-1t substrate at 37°C (lower band: unreacted ribozyme; upper band: ribozyme reacted with S-1t substrate). Summarized data from all assays is shown in Table 1.