Oxazolidinone antibiotics target the P site on Escherichia coli ribosomes

Abstract

The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

FIG. 1.

Structures of oxazolidinones PNU-100766 (I), PNU-140693 (II), and PNU-176798 (III).

FIG. 2.

Effect of oxazolidinone PNU-176798 on the dissociation of initiation complexes. The initiation complexes were formed as described in Materials and Methods and were undiluted (lanes 1 and 2) or diluted 10-fold (lanes 3 and 4) or 20-fold (lanes 5 and 6) in buffer A containing 6 mM Mg(Ac)₂ in the presence of 80 μM PNU-176798 (lanes 2, 4, and 6) or in its absence (lanes 1, 3, and 5). Reactions were continued for 5 min at 35°C. The f[³⁵S]Met-tRNA that remained bound to the ribosomes was then measured by filtration on nitrocellulose filters. Determinations were performed in duplicate.

FIG. 3.

Lineweaver-Burke plots of the inhibition by oxazolidinone PNU-176798 of the fMet-puromycin formation. Formation of fMet-puromycin was measured as described in Materials and Methods, by using different concentrations of f[³⁵S]Met-tRNA. Each reaction mixture contained the following concentrations of the antibiotic PNU-176798: •, no antibiotic; ▪, 16.6 μM; ▴, 50.0 μM; ▾, 66 μM.

FIG. 4.

Effect of oxazolidinone PNU-100766 on peptide bond formation using the fragment reaction. The reactions were conducted at 4°C as described by Monroe (21), and the reaction mixtures contained 60 mM MgCl₂, 1 mM puromycin, 33% ethanol, and 50 pmol 70S or 50S subunits. Determinations were performed in duplicate.

FIG. 5.

Effect of oxazolidinone PNU-176798 on EF-P-stimulated synthesis of peptide bonds. Reactions were conducted with 15% methanol, 1 μM puromycin, and EF-P as described in Materials and Methods. The antibiotic was added in the first incubation during formation of the initiation complex. □, reaction mixtures without added EF-P. Where indicated, 0.4 μg of EF-P was added to each reaction mixture (▪).

FIG. 6.

Preferential effect of oxazolidinone PNU-176798 on the initiation step preceding peptide bond synthesis. Initiation complex formation was allowed to proceed for 20 min at 30°C with the indicated concentrations of the antibiotic, and then 1 μM puromycin and 0.4 μg of EF-P were added (⋄). The antibiotic was added after formation of the initiation complex prior to the addition of puromycin and EF-P (□).

FIG. 7.

Effect of EF-G on the translocation of fMet-tRNA from the ribosomal A site to the P site. Initiation complexes were formed as described in Materials and Methods. The reactions were performed at 0°C and, where indicated, contained 1.5 pmol of EF-G and/or the designated concentrations of the oxazolidinone PNU-176798. (A) Reactions in the presence (column 1) or absence (column 2) of EF-G. (B) Oxazolidinone concentration required to inhibit EF-G-dependent translocation. The f[³⁵S]Met-puromycin product was measured by ethyl acetate extraction as described in Materials and Methods. Symbols: □, reaction mixtures containing EF-G; ⋄, reaction mixtures without EF-G.