Aminoglycosides suppress tRNA processing in human epidermal keratinocytes in vitro.

The ever-growing resistance of pathogens to antibiotics and the lack of potent antibacterial drugs constitute major problems in the treatment of infectious diseases. Thus, the better understanding of the mode of action of antibiotics at the molecular level is of essential importance. Accumulating evidence points towards RNA as being a crucial target of antibacterial and antiviral drugs. Interestingly, aminoglycosides, one of the most important families of antibiotics, apart from their inhibitory effect on ribosome function, reportedly interfere with various RNA molecules and in vitro suppress the proliferation of human keratinocytes. In this study we investigated the effect of the aminoglycosides neomycin B, paromomycin, tobramycin and gentamycin on ribonuclease P activity from normal human epidermal keratinocytes. All aminoglycosides tested revealed a dose-dependent inhibition of tRNA maturation, which was reduced by increasing Mg(2+) ion concentrations, indicating competition of the cationic aminoglycosides with magnesium ions required for catalysis. Our in vitro findings suggest that the inhibitory effects of aminoglycosides on tRNA processing may be implicated in the mechanisms of their antiproliferative action on human epidermal keratinocytes.

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

Effect of peptidyltransferase inhibitors on ribonuclease P activity from Dictyostelium discoideum. Effect of antibiotics on RNase P.

The effect of several peptidyltransferase inhibitors on ribonuclease P activity from Dictyostelium discoideum was investigated. Among the inhibitors tested puromycin, amicetin and blasticidin S revealed a dose-dependent inhibition of tRNA maturation. Blasticidin S and amicetin do not compete with puromycin for the same site on the enzyme, suggesting the existence of distinct antibiotic binding sites on D. discoideum RNase P. Inhibition experiments further indicate that binding sites for blasticidin S and amicetin overlap.

Inhibition of eukaryotic ribonuclease P activity by aminoglycosides: kinetic studies.

The effect of several aminoglycoside antibiotics on ribonuclease P (RNase P) was investigated using an in vitro experimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, paromomycin, neomycin) behave as classical non-competitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic competition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg(2+) ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides compete with Mg(2+) for common binding sites on RNase P holoenzyme.

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity.

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.