Insights into the bottromycin A ₂ mechanism of action

The spread of antimicrobial resistance among pathogenic bacteria poses a threat for modern medicine, highlighting the need for the discovery and development of new potential therapeutic agents. Bottromycin A ₂ (BotA2) represents a promising candidate for future drug development, exhibiting activity against clinically relevant methicillin-resistant Staphylococcus aureus , vancomycin-resistant Enterococcus , and mycoplasma. However, its exact mechanism of action has not been fully elucidated until now. Here, we demonstrate that BotA2 inhibits bacterial translation showing unique context specificity with regard to the mRNA coding sequence. By using high-throughput toe-printing combined with deep sequencing (toe-seq analysis), we show that BotA2 induces ribosome stalling predominantly when a glycine codon enters the A-site of the ribosome, with stalling efficiency independent of codons located in the P- and E-sites. Our biochemical and biophysical data reveal that BotA2 arrests glycine-containing ternary complexes on the ribosome thereby preventing the full accommodation of incoming Gly-tRNA ^Gly in the peptidyl transferase center. Altogether, our findings uncover a completely novel, previously undescribed mechanism of translation inhibition based on the context-specific immobilization of ternary complexes on elongating ribosomes.