Seefeldt, A. C. | bioRxiv Science

bioRxiv2026

Ribosome collisions trigger tmRNA-mediated rescue through mRNA disengagement

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{middle dot}peptidyl-tRNA complexes and shows that Hsp15, previously thought to interact exclusively with 50S{middle dot}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.

molecular biology↗

bioRxiv2019

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1-3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in {gamma}-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

microbiology↗