The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu.

Abstract

It was previously shown that, in cell-free systems, virginiamycin M blocks the elongation of polypeptide chains by inhibiting both the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu (EF-Tu) and the peptidyl transferase reaction. The present work aims at identifying the reaction primarily affected by this antibiotic. Ribosomes, whether treated or not with virginiamycin M, are able to activate the EF-Tu GTPase under conditions in which the reaction takes place uncoupled from the binding of aminoacyl-tRNA to ribosomes. Since this antibiotic has no inhibitory action on the elongation factor G (EF-G) GTPase either, it can be concluded that the ribosomal centers for elongation factor GTPases are conclusively not affected by virginiamycin M. In the presence of kirromycin, an antibiotic that prevents dissociation of EF-Tu from the ribosome after GTP hydrolysis, virginiamycin M does not inhibit the enzymatic binding of Phe-tRNA to the poly(U)-ribosome complex or does it affect the concomitant stoichiometric hydrolysis of GTP. Under these conditions, ribosomal complexes carrying Phe-tRNA, kirromycin, EF-Tu and GDP at the A site are formed as well in the presence as in the absence of virginiamycin M. Moreover, the release of EF-Tu from ribosomes incubated with virginiamycin M was found to occur normally in the absence of kirromycin, thus suggesting an interference by the former inhibitor with an elongation step that follows the detachment of EF-Tu - GDP from the ribosomes. Indeed, aminoacyl-tRNA becomes sensitive to virginiamycin M after the release of EF-Tu, as the addition of the inhibitor at this stage promotes the release of Phe-tRNA bound enzymatically to the A site. Consequently, the accumulation of ribosomal complexes carrying an aminoacyl-tRNA at the A site is prevented, and this mimics an inhibition of the binding reaction. On the other hand, such a dissociation induced by virginiamycin M is expected to regenerate ribosomes with an empty A site, and to allow an additional round of the binding process on the same ribosome in the absence of translocation. Accordingly, the GTP hydrolysis coupled with the EF-Tu-directed binding of Phe-tRNA to the poly(U) · ribosome complex was found markedly stimulated by virginiamycin M. These observations can be accounted for by postulating that virginiamycin M acts primarily on the peptidyl transferase: the release of aminoacyl-tRNA from the A site is, then, due to an inability of the CCA-aminoacyl end of tRNA to bind to the peptidyl transferase center inactivated by the antibiotic.