Drug Sensing by the Ribosome Induces Translational Arrest via Active Site Perturbation

Abstract

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.

The general mode of translation inhibition by macrolide antibiotics

“…Recent structural studies revealed that out of the C-terminal amino acids of the stalled nascent peptide, residues −8 to −2 (if the C-terminal residue is assigned position number 0) are in close proximity to the NPET-bound macrolide antibiotic (14,20). Consequently, we anticipated that this segment of the nascent polypeptide chain that interacts with the drug would define the sites of macrolide-induced ribosome stalling.…”

Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

“…To determine the structures of EVN and AVI on the ribosome, we prepared Erm-stalled ribosome complexes (SRCs) as reported (25,26). The SRCs were incubated with either 100 μM EVN or AVI, and the complexes were then subjected to singleparticle cryo-EM analysis (Materials and Methods).…”

“…The SRCs were prepared essentially as described (25,44). Cryo-EM data collection was performed on the Titan Krios (FEI) 300-kV TEM equipped with a Falcon II direct electron detector.…”

“…Images of individual ribosome particles were aligned by using Motion Correction software (45), and then particles were selected automatically by using SIGNATURE (46). All images were processed by using a frequency-limited refinement protocol that prevents overfitting (47) using the SPIDER software package (48), as described (25,44). The final maps were subjected to the program EM-BFACTOR (49) to apply an automatically determined negative B factor for sharpening of the map, and local resolution was calculated by using ResMap (50).…”

Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

“…They are integral parts of regulatory nascent polypeptides that monitor cellular physiology by undergoing regulated translational arrest (10,11). Arrest sequences interact with the ribosomal components at the peptidyl transferase center and/or the exit tunnel to interfere with the peptidyl transfer, translocation, or termination functions of the ribosome (12)(13)(14)(15). They are diverse in length and primary sequences; translation arrest is either inducible with a specific small molecule or is intrinsic but is subject to release by a physical pulling force that is applied to the nascent chain (10,16,17).…”