Sequence-Dependent Elongation Dynamics on Macrolide-Bound Ribosomes

•Erythromycin-induced ribosome stalling and pausing are abrupt events•Elongation rates are not perturbed in general by NPET-bound erythromycin•The ermCL-stalled ribosome has a selective A site•Synthesis of readthrough peptides involves a pause in the rotated ribosomal state

SummaryThe traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

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