Cis-acting resistance peptides reveal dual ribosome inhibitory action of the macrolide josamycin

Macrolide antibiotics block the entrance of nascent peptides to the peptide exit tunnel of the large ribosomal subunit. Expression of specific cis-acting peptides confers low-level macrolide-resistance. We show that, in the case of josamycin, peptide expression does not eject josamycin from the ribosome, implying a peptide resistance mechanism different from that previously suggested for erythromycin. We find dipeptide formation and dipeptidyl-tRNA drop-off in the presence of josamycin to be much slower during translation of resistance than of control mRNAs. We demonstrate low-level josamycin resistance by over-expression of peptidyl-tRNA hydrolase. These findings suggest dual growth-inhibitory action of josamycin by (i) direct inhibition of peptide-elongation and (ii) indirect inhibition of peptide-elongation through rapid peptidyl-tRNA drop-off, leading to depletion of tRNA isoacceptors available for protein synthesis. We propose that josamycin resistance peptide expression brings ribosomes into a “quarantine” state with small drop-off rate, thereby eliminating the josamycin dependent depletion of tRNA isoacceptors in the protein-synthesis-active state.