Nunez, E.; Saha, P.; G.Ibarluzea, M.; Muguruza-Montero, A.; M.Alicante, S.; Ramis, R.; Leonardo, A.; Bergara, A.; Villarroel, A.; Rico, F.

Trigger Factor (TF) is an ATP-independent chaperone that assists in co-translational protein folding by associating with ribosomes to prevent aggregation. While its interaction with ribosomes has been described, real-time visualization of TF dynamics has remained elusive. Using high-speed atomic force microscopy, we imaged full 70S ribosomes under near-physiological conditions during translation. TF exhibited dynamic transitions between extended and compact conformations, forming both stable and transient contacts with ribosomal proteins uL23 and bL17 in ribosome nascent chain complexes. Binding to non-translating ribosomes was not observed under these conditions. Molecular dynamics simulations of TF alone and in complex with ribosomal proteins supported the experimental observations. Our findings reveal the structural flexibility of TF and its selective association with active ribosomes. Our combination of experimental and computational approaches offers new insights into how TF dynamically engages ribosomes during translation to facilitate protein folding.