Central to the transcriptional control of the Escherichia coli heat shock regulon is the stress-dependent inhibition of the σ³² subunit of RNA polymerase by reversible association with the DnaK chaperone, mediated by the DnaJ cochaperone. Here we identified two distinct sites in σ³² as binding sites for DnaK and DnaJ. DnaJ binding destabilizes a distant region of σ³² in close spatial vicinity of the DnaK-binding site, and DnaK destabilizes a region in the N-terminal domain, the primary target for the FtsH protease, which degrades σ³² in vivo. Our findings suggest a molecular mechanism for the DnaK- and DnaJ-mediated inactivation of σ³² as part of the heat shock response. They furthermore demonstrate that DnaK and DnaJ binding can induce conformational changes in a native protein substrate even at distant sites, a feature that we propose to be of general relevance for the action of Hsp70 chaperone systems.