Metelev, M.; Borg, A.; Akbar, S.; Larsson, D. S. D.; Seefeldt, A. C.; Selmer, M.; Johansson, M.

Ribosome rescue pathways maintain translation during normal growth and contribute to stress tolerance. In Escherichia coli, HrpA is a ribosome-collision recognition factor that mediates antibiotic tolerance by resolving stalled ribosomes, yet the fate of the resulting ribosomal intermediate, with a topologically trapped peptidyl-tRNA in the 50S nascent peptide exit tunnel, has remained unclear. Using in vivo single-molecule tracking, we show that under antibiotic treatment, HrpA-dependent collision processing promotes recruitment of tmRNA and Hsp15 as downstream rescue factors. Cryo-electron microscopy of affinity-purified native complexes from tmRNA-deficient cells reveals accumulation of aberrant 70S*peptidyl-tRNA complexes and shows that Hsp15, previously thought to interact exclusively with 50S*peptidyl-tRNA, binds to 70S complexes lacking mRNA in the E site. This intermediate can be resolved by the canonical tmRNA pathway, thereby connecting HrpA-dependent collision processing to canonical tmRNA-mediated rescue.