Oxazolidinone Antibiotics Target the P Site onEscherichiacoliRibosomes

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 fMettRNA 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.A novel class of antimicrobial agents, the oxazolidinones, target a wide spectrum of gram-positive and anaerobic bacteria (4, 6, 9, 28). These compounds act by inhibiting protein synthesis and have no effect on replication or transcription (8). Cell extracts exposed to oxazolidinones are impaired in protein synthesis when programmed by native mRNAs but do not appear to be inhibited when programmed by synthetic mRNAs that lack the signals required for initiation and termination of translation (7,8,26). This suggested that these compounds may target the initiation reaction. The oxazolidinone PNU-100766 (linezolid; Fig. 1) inhibits binding of the initiator fMettRNA to the 70S ribosomal particle programmed with a synthetic mRNA that harbors a Shine-Dalgarno sequence and a properly spaced initiation codon (29).Mutations to oxazolidinone resistance map to domain V of the 23S rRNA in Halobacterium halobium (18), Staphylococcus aureus (27), and the enterococci while mapping to domains IV and V in Escherichia coli (33). The position of these PNU-100766 resistance mutations suggested to us that the oxazolidinones may target peptidyl transferase indirectly by affecting the binding of the initiator tRNA. Since the P site accommodates the initiator tRNA and the nascent protein, these drugs could also affect the affinity of the peptidyl-tRNA for the ribosome.Recent studies have indicated that the oxazolidinones bind to 70S ribosomes, as well as to 50S subunits (19), but not to 30S subunits. In contrast, a report by Matassova et al. (20) demonstrated that oxazolidinone footprints map to the central domain of the 16S rRNA whereas the 23S rRNA ...

Cross-linking in the Living Cell Locates the Site of Action of Oxazolidinone Antibiotics

“…Consistent with this model, cross-linking of tRNA was observed in our studies, suggesting that occupation of one of the tRNA sites on the ribosome may facilitate drug binding. Several studies demonstrate that oxazolidinones may interfere with positioning of fMet-tRNA in the P-site of 70 S ribosomes, thereby preventing the initiation of translation (8,16,18). In agreement with this conclusion, the A-2602 base that is exclusively cross-linked by 125 I-XL in the living cell has been implicated in the interaction of the 3Ј end of transfer RNA with the ribosome (56).…”

“…In addition, these studies conducted with isolated ribosomes had pointed to possible interactions of oxazolidinones with A-864 in the 16 S rRNA of the small ribosomal subunit. Lack of a clear understanding of the oxazolidinone binding site on the ribosome and reliance on indirect approaches led to the proposal of diverse models of oxazolidinone action (7,8,9,12,(15)(16)(17)(18)(19).…”

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning

“…Because the drug does not encroach on the P site (Fig. 4F), we suggest that the differential effects that oxazolidinones have on fMet-tRNA binding at the P site (11,19,22,32) are probably indirect and related to the nonproductive drug-induced conformation of nucleotides within the PTC, such as U2585 and U2506.…”

“…4F). Thus, it is unclear why in some systems oxazolidinones have little inhibitory effect on fMetpuromycin formation (23,31,32), whereas in others an obvious effect is observed (15,20,22). Because the drug does not encroach on the P site (Fig.…”

Targeting the A site RNA of the Escherichia coli ribosomal 30 S subunit by 2′-O-methyl oligoribonucleotides: a quantitative equilibrium dialysis binding assay and differential effects of aminoglycoside antibiotics

“…Other antibiotics have been found to bind 23 S rRNA at the peptidyl transferase centre and inhibit protein synthesis, either directly by preventing peptide-bond formation (chloramphenicol and clindamycin) or indirectly by causing dissociation of peptidyl tRNA (macrolides) [5][6][7]. Oxazolidinone antibiotics inhibit the initiation process, probably by competing with the fMet (formylmethionine) tRNA for the binding to the peptidyl transferase centre of the 23 S rRNA [8,9], whereas tetracycline binds 16 S rRNA and inhibits decoding [10].…”