Inducible expression of the erm erythromycin resistance genes relies on drug-dependent ribosome stalling. The molecular mechanisms underlying stalling are unknown. We used a cell-free translation system to elucidate the contribution of the nascent peptide, the drug, and the ribosome toward formation of the stalled complex during translation of the ermC leader cistron. Toe-printing mapping, selective amino acid labeling, and mutational analyses revealed the peptidyl transferase center (PTC) as the focal point of the stalling mechanism. In the ribosome exit tunnel, the C-terminal sequence of the nascent peptide, critical for stalling, is in the immediate vicinity of the universally conserved A2062 of 23S rRNA. Mutations of this nucleotide eliminate stalling. Because A2062 is located in the tunnel, it may trigger a conformational change in the PTC, responding to the presence of a specific nascent peptide. The cladinose-containing macrolide antibiotic in the tunnel positions the nascent peptide for interaction with the tunnel sensory elements.

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.

In bacteria, ribosome-stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase center (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural bases for understanding how the drug and the nascent peptide cooperate to inhibit peptide-bond formation and induce translation arrest.

Macrolide antibiotics inhibit protein synthesis by targeting the bacterial ribosome. They bind at the nascent peptide exit tunnel and partially occlude it. Thus, macrolides have been viewed as 'tunnel plugs' that stop the synthesis of every protein. More recent evidence, however, demonstrates that macrolides selectively inhibit the translation of a subset of cellular proteins, and that their action crucially depends on the nascent protein sequence and on the antibiotic structure. Therefore, macrolides emerge as modulators of translation rather than as global inhibitors of protein synthesis. The context-specific action of macrolides is the basis for regulating the expression of resistance genes. Understanding the details of the mechanism of macrolide action may inform rational design of new drugs and unveil important principles of translation regulation.

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.

SUMMARY During protein synthesis, nascent polypeptide chains within the ribosomal tunnel can act in cis to induce ribosome stalling and regulate expression of downstream genes. The Staphylococcus aureus ErmCL leader peptide induces stalling in the presence of clinically important macrolide antibiotics, such as erythromycin, leading to the induction of the downstream macrolide resistance methyltransferase ErmC. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmCL-stalled ribosome at 3.9 Å resolution. The structure reveals how the ErmCL nascent chain directly senses the presence of the tunnel-bound drug and thereby induces allosteric conformational rearrangements at the peptidyltransferase center (PTC) of the ribosome. ErmCL-induced perturbations of the PTC prevent stable binding and accommodation of the aminoacyl-tRNA at the A-site leading to inhibition of peptide bond formation and translation arrest.

Bacterial populations contain persisters, cells which survive exposure to bactericidal antibiotics and other lethal factors. Persisters do not have a genetic resistance mechanism, and their means to tolerate killing remain unknown. In exponentially growing populations of Escherichia coli the frequency of persister formation usually is 10 ؊7 to 10 ؊5 . It has been shown that cells overexpressing either of the toxic proteins HipA and RelE, both members of the bacterial toxin-antitoxin (TA) modules, have the ability to form more persisters, suggesting a specific role for these toxins in the mechanism of persistence. However, here we show that cells expressing proteins that are unrelated to TA modules but which become toxic when ectopically expressed, chaperone DnaJ and protein PmrC of Salmonella enterica, also form 100-to 1,000-fold more persisters. Thus, persistence is linked not only to toxicity caused by expression of HipA or dedicated toxins but also to expression of other unrelated proteins.

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