Fig. 1: A fluorescent-based reporter system for protein abundance and DnaK response.
From: DnaK response to expression of protein mutants is dependent on translation rate and stability
a Overview of reporter system: (1) The addition of IPTG allows for transcription of the GOI (gene of interest)-tagRFP by RNA polymerase in a complex with the transcription factor σ70. (2) Following transcription of GOI-tagRFP the protein of interest, POI-tagRFP is synthesised. (3) DnaK releases the transcription factor σ32 and binds to unfolded/misfolded POI-tagRFP. (4) σ32 binds to RNA polymerase. (5) Binding of σ32 to RNA polymerase allows for transcription of endogenous PQC proteins, such as DnaK. The RNA polymerase—σ32 complex will also bind to the DnaK promoter on the reporter plasmid and transcribe the sfGFP gene. (6) Following transcription of the sfGFP gene, the protein sfGFP will be synthesised. b The N-terminal domain of the λ repressor (blue) bound to DNA (purple). Mutated residues are shown as sticks (blue). PDB:1lmb56.
