Functional dissection of Trigger factor and DnaK: Interactions with nascent polypeptides and thermally denatured proteins

In Escherichia coli, the ribosome-associated Trigger Factor (TF) cooperates with the DnaK system in the folding of newly synthesized cytosolic polypep tides. Here we investigated the functional relationship of TF and DnaK by c omparing various functional properties of both chaperones. First, we analyz ed the ability of TF and DnaK to associate with nascent polypeptides and fu ll-length proteins released from the ribosome. Toward this end, we establis hed an E. coli based transcription/translation system containing physiologi cal ratios of TF, DnaK and ribosomes. In this system, TF can be crosslinked to nascent polypeptides of sigma (32). No TF crosslink was found to full-l ength sigma (32), which is known to be a DnaK substrate. In contrast, DnaK crosslinked to both nascent and full-length sigma (32). DnaK crosslinks cri tically depended on the type of chemical crosslinker. Crosslinks represent specific substrate-chaperone interactions since they relied on the associat ion of the nascent polypeptides with the substrate binding pocket of DnaK. While DnaK is known to be the major chaperone to prevent protein aggregatio n under heat shock conditions, we found that TF did not prevent aggregation of thermally unfolded proteins in vitro and was not able to complement the heat-sensitive phenotype of a Delta dnaK52 mutant in vivo. These data indi cate that TF and DnaK show strong differences in their ability to prevent a ggregation of denatured proteins and to associate with native like substrat es, but share the ability to associate with nascent polypeptides.