Oh, E. et al. Cell 147, 1295–1308 (2011).

Ribosome profiling, a method in which ribosome-protected fragments of RNA are deep-sequenced, is helping to answer what happens as nascent polypeptides leave the ribosome in eukaryotic cells. Oh and colleagues now capture bacterial ribosomes in the act of translation by chloramphenicol treatment or fast filtration of culture. Polysome purification and micrococcal nuclease digestion produces monosomes, and then the researchers reverse-transcribe and sequence the ribosome-protected footprints. The researchers profile nascent-chain interactions with the chaperone trigger factor by chemically cross-linking to stabilize transient interactions and selectively purifying the ribosomal fraction associated with the protein. They show that trigger factor associates with nascent peptide 100 amino acids beyond the ribosomal exit site, allowing other proteins to access the newest parts of the polypeptide. This is in contrast to in vitro studies that indicated immediate surveillance by trigger factor.