A Unique Mechanism of Glycine-Specific Inhibition of Bacterial Translation by Bottromycin A₂

The rise of antimicrobial resistance among pathogenic bacteria poses a critical challenge to modern medicine, highlighting an urgent need for novel therapeutic agents. Bottromycin A₂ (BotA2) is a promising candidate for future drug development, demonstrating potent activity against clinically relevant pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Mycoplasma species, although its molecular mechanism of action has remained unclear until now. Here, we demonstrate that BotA2 inhibits bacterial translation with unique context specificity determined by the mRNA coding sequence. Using high-throughput toe-printing coupled with deep sequencing (Toe-seq analysis), we show that BotA2 induces ribosome pausing predominantly when a glycine codon enters the A-site of the ribosome, regardless of the codon identities in the P- and E-sites. Our biochemical and biophysical data indicate that BotA2 specifically arrests glycine-delivering ternary complexes on the ribosome, thereby preventing full accommodation of incoming Gly-tRNA^Gly within the peptidyl transferase center. Altogether, our findings uncover a previously undescribed mechanism of translation inhibition, driven by the context-specific immobilization of ternary complexes on elongating ribosomes.