Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization, and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNAfMet enable selective site-specific labeling of nascent proteins at N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time ca. 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are only detected for a sequence encoding a globular protein but not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multi-component, severely rotationally restricted and strongly chain length/sequence-dependent nascent chain dynamics.

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