Pentamidine inhibits catalytic activity of group I intron Ca.LSU by altering RNA folding

Abstract

The antimicrobial agent pentamidine inhibits the self-splicing of the group I intron Ca.LSU from the transcripts of the 26S rRNA gene of Candida albicans, but the mechanism of pentamidine inhibition is not clear. We show that preincubation of the ribozyme with pentamidine enhances the inhibitory effect of the drug and alters the folding of the ribozyme in a pattern varying with drug concentration. Pentamidine at 25 microM prevents formation of the catalytically active F band conformation of the precursor RNA and alters the ribonuclease T1 cleavage pattern of Ca.LSU RNA. The effects on cleavage suggest that pentamidine mainly binds to specific sites in or near asymmetric loops of helices P2 and P2.1 on the ribozyme, as well as to the tetraloop of P9.2 and the loosely paired helix P9, resulting in an altered structure of helix P7, which contains the active site. Positively charged molecules antagonize pentamidine inhibition of catalysis and relieve the drug effect on ribozyme folding, suggesting that pentamidine binds to a magnesium binding site(s) of the ribozyme to exert its inhibitory effect.

Figure 1

Preincubation of Ca.LSU precursor with pentamidine enhances its inhibitory effect on splicing. Equal amounts of radiolabeled Ca.LSU precursor RNA were added to each reaction. The non-preincubated reactions were run under standard splicing conditions with the indicated concentrations of pentamidine (lanes 1–5). The other samples were preincubated in Tris buffer at 37°C for 20 min before addition of MgCl₂, spermidine and GTP. The indicated concentrations of pentamidine were added either during preincubation (lanes 6–10) or after preincubation together with the other splicing components (lanes 11–16) and reaction products were analyzed by PAGE as described in Materials and Methods. The asterisks indicate products possibly generated by Mg²⁺-induced hydrolysis of the Ca.LSU precursor RNA.

Figure 2

Kinetics of pentamidine inhibition of splicing by Ca.LSU. (A) Indicated concentrations of pentamidine were added to the splicing reaction together with all splicing components without preincubation of the precursor RNA (Materials and Methods). (B) The indicated concentrations of pentamidine were present during a 20 min preincubation of precursor RNA and splicing was initiated by addition of all other splicing components. In both panels the time course of the splicing reaction was analyzed as in Figure 1. All experiments were performed four times and the average splicing fraction [the ratio of spliced intron (I) and ligated exons (E1–E2) to total RNA] is presented (standard deviations indicated).

Figure 3

Pentamidine alters the folding of Ca.LSU precursor RNA. The same amount of radiolabeled Ca.LSU precursor in Tris buffer was incubated at 0 or 37°C for 20 min in the presence of the indicated concentration of pentamidine and was then analyzed by native PAGE (Materials and Methods). The two different exposures were to allow clear visualization of the S and F bands (Exposure 1) and smear RNA species (Exposure 2).

Figure 4

Pentamidine alters the folding of Ca.LSU precursor RNA. The same amount of radiolabeled Ca.LSU precursor in Tris buffer was incubated at 0 or 37°C for 20 min in the presence of the indicated concentration of pentamidine and was then analyzed by native PAGE (Materials and Methods). The two different exposures were to allow clear visualization of the S and F bands (Exposure 1) and smear RNA species (Exposure 2).

Figure 4

Pentamidine alters the folding of Ca.LSU precursor RNA. The same amount of radiolabeled Ca.LSU precursor in Tris buffer was incubated at 0 or 37°C for 20 min in the presence of the indicated concentration of pentamidine and was then analyzed by native PAGE (Materials and Methods). The two different exposures were to allow clear visualization of the S and F bands (Exposure 1) and smear RNA species (Exposure 2).

Figure 5

Mg²⁺ and spermidine reduce pentamidine inhibition of Ca.LSU catalysis. Equal amounts of radiolabeled Ca.LSU precursor were either assayed for ribozyme activity without preincubation in the presence of varying levels of pentamidine or preincubated before ribozyme assays in buffer alone or in the presence of 1.25 mM MgCl₂, 10 µM GTP or 0.4 mM spermidine. All assays with and without preincubation were performed under standard splicing conditions. Lane P is a sample preincubated in buffer alone without adding splicing components (magnesium, spermidine, GTP).

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).

Figure 7

Positively charged molecules antagonize pentamidine inhibition directly in the splicing reaction. Equal amounts of radiolabeled Ca.LSU precursor RNA were spliced in the presence of the indicated concentrations of pentamidine and MgCl₂ (A) or spermidine (B) at 37°C for 20 min. Only the concentrations of the indicated splicing component varied, while the other splicing components remained as in the standard splicing reaction. The fraction pentamidine resistance (Fr) is defined as in Figure 6.

Figure 8

Positively charged molecules eliminate the pentamidine-induced alteration in ribonuclease T1 sensitivity. Equal amounts of 5′ end radiolabeled intron RNA was preincubated in Tris–HCl in the presence of 0, 25 or 100 µM pentamidine and 1.25 mM MgCl₂, 1.25 mM CaCl₂ or 0.4 mM spermidine at 37°C for 20 min. Then ribonuclease T1 cleavage and PAGE were performed as described in Materials and Methods. The major cleaved positions are indicated at the left of the gel (nucleotide numbers as in Figure 4).

Figure 9

MgCl₂ antagonizes the alteration of folding of Ca.LSU precursor RNA induced by pentamidine. Equal amounts of radiolabeled precursor RNA were preincubated in Tris–HCl in the presence of 0, 25 or 100 µM pentamidine and in the absence or presence of 1.25 mM MgCl₂, 1.25 mM CaCl₂ or 0.4 mM spermidine at 37°C for 20 min, then analyzed by native PAGE as described in Materials and Methods.