Figure 6.

Dose-dependent effect of positively charged molecules on ribozyme sensitivity to pentamidine. Equal amounts of radiolabeled Ca.LSU precursor RNA were preincubated in the presence or absence of 100 µM pentamidine with the indicated concentrations of CaCl₂, MgCl₂ or spermidine at 37°C for 20 min. The missing splicing reaction components (and varying concentrations of CaCl₂, MgCl₂ or spermidine) were then added. However, in the subsequent splicing reactions, divalent cation concentrations were 1.25 mM MgCl₂ and 1.25 mM CaCl₂ for experiments in which CaCl₂ was present during preincubation and 2.5 mM MgCl₂ when MgCl₂ was present in the preincubation (A), and 2.5 mM MgCl₂ when spermidine (B) was present in the preincubation. Spermidine concentration was 0.4 mM in all splicing reactions. All data were obtained from triplicate experiments. Each gel was scanned and analyzed by PhosphorImager and the splicing percentage was obtained for each reaction. The fraction pentamidine resistance (Fr) represents the ratio of Ca.LSU splicing activity of the reaction including pentamidine to that of the pentamidine-free reaction at each cation concentration. These data were plotted using the program PRISM v.2.01 (GraphPad Software, San Diego, CA).