Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains

Area of Science:

• Molecular Biology
• Protein Folding
• Bacterial Physiology

Background:

• The role of Escherichia coli Hsp70 (DnaK) in de novo protein folding under non-stress conditions has been unclear.
• Trigger factor is a ribosome-associated chaperone in E. coli with a known role in early protein folding.

Purpose of the Study:

• To investigate the involvement of DnaK in chaperoning nascent polypeptides during de novo protein folding in E. coli.
• To elucidate the functional relationship and potential overlap between DnaK and trigger factor in maintaining cellular viability.

Main Methods:

• Utilized immunoprecipitation assays to detect interactions between DnaK and nascent polypeptides.
• Generated single and double deletion mutants for DnaK and trigger factor genes.
• Assessed bacterial growth and viability under normal conditions for the generated mutants.

Main Results:

• DnaK was found to transiently associate with a diverse range of newly synthesized proteins, particularly those larger than 30 kDa, under non-stress conditions.
• Deletion of the trigger factor gene led to a significant increase (doubling) in the association of DnaK with nascent polypeptides.
• Simultaneous deletion of both DnaK and trigger factor genes resulted in lethality in E. coli under standard growth conditions.

Conclusions:

• DnaK plays a significant role in the de novo folding of nascent polypeptides, even in the absence of stress.
• DnaK and trigger factor possess important, partially overlapping functions in protein folding.
• The observed overlapping functions explain the previously noted E. coli's tolerance to the loss of either DnaK or trigger factor individually.