The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria

Abstract

The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [3H]N-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli. In addition, complex formation with Staphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA to E. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2-N-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of the N-formylmethionyl-tRNA-ribosome-mRNA ternary complex.

FIG. 1

(A) Linezolid inhibition of E. coli initiation complexes formed in the presence of IF1, IF2, IF3, and the defined mRNA described in Materials and Methods. Either 30S (•) or 70S (□) initiation complexes were formed by using stoichiometric amounts of each initiation factor and the appropriate ribosomal subunit. The complexes were allowed to form for 10 min at 37°C. (B) Kasugamycin inhibition of E. coli 70S initiation complex formation.

FIG. 2

Inhibition of S. aureus 70S translation initiation complex formation by linezolid. [³H]tRNA^fMet binding to 70S ribosomal subunits was measured in the presence of the defined oligoribonucleotide described in Materials and Methods. Complexes were allowed to form for 10 min at 37°C.

FIG. 3

Lack of effect of linezolid on mRNA binding to ribosomes. (A) Purified E. coli 70S ribosomes and [³²P]mRNA (defined sequence of 200 bp) were incubated for 15 min at 24°C in the presence or absence of 200 μM linezolid (Lin) before trapping the complex on nitrocellulose filters.

FIG. 4

Effect of linezolid on E. coli 70S initiation complexes formed in the absence of initiation factors with the triplet codon AUG used as the source of mRNA. Initiation complex formation was as described in Materials and Methods.