Macrolide antibiotics allosterically predispose the ribosome for translation arrest

Significance Translation arrest regulated by nascent peptides and small cofactors controls expression of important genes, including medically relevant macrolide antibiotic resistance genes. The role of the cofactor for triggering this mechanism has remained enigmatic. Previous studies suggested that extensive interactions between the nascent chain and the antibiotic molecule juxtaposed in the ribosomal exit tunnel were critical for halting translation. However, here we show that the antibiotic induces stalling, even without significant contacts with the peptide, by allosterically altering the peptidyl transferase center. This finding unveils a previously unknown role of cofactors for translation arrest and demonstrates the existence of a functional link between the exit tunnel and the catalytic center of the ribosome. Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.