Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factors 1 or 2 subsequent to release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with release factor 1 and Api137 reveals the molecular interactions that lead to release factor trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons prior to polypeptide release, thereby resulting in a global shutdown of translation termination.

The ribosome is able to monitor the structure of the nascent peptide and can stall in response to specific peptide sequences. Such programmed stalling is used for the regulation of gene expression. The molecular mechanisms of the nascent‐peptide recognition and ribosome stalling are unknown. We identified the conserved and posttranscriptionally modified 23S rRNA nucleotide m2A2503 located at the entrance of the ribosome exit tunnel as a key component of the ribosomal response mechanism. A2503 mutations abolish nascent‐peptide‐dependent stalling at the leader cistrons of several inducible antibiotic resistance genes and at the secM regulatory gene. Remarkably, lack of the C2 methylation of A2503 significantly function induction of expression of the ermC gene, indicating that the functional role of posttranscriptional modification is to fine‐tune ribosome–nascent peptide interactions. Structural and biochemical evidence suggest that m2A2503 may act in concert with the previously identified nascent‐peptide sensor, A2062, in the ribosome exit tunnel to relay the stalling signal to the peptidyl transferase centre.

Highlights d Retapamulin arrests bacterial ribosomes specifically at translation start sites d Retapamulin-assisted Ribo-seq reveals known and cryptic translation start sites d Translation from start sites located within the ORFs may generate functional proteins d Start-stop sites found within some genes may help to regulate gene expression

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.ribosome | antibiotics | protein synthesis | nascent peptide | oxazolidinones