The shape of the ribosome exit tunnel affects cotranslational protein folding.

The Escherichia coli ribosome exit tunnel can accommodate small folded proteins, while larger ones tend to fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using ribosomes with deletions in proteins uL23 and uL24, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain folding close to the uL23 loop in wildtype ribosomes folds deeper in the tunnel in uL23 Δloop (but not in uL24 Δloop) ribosomes, while two ~100-residue domains that fold close to the uL24 loop near the tunnel exit in wildtype ribosomes fold at deeper locations in uL24 Δloop (but not in uL23 Δloop) ribosomes. These results provide strong support to the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.