BOTH AMBIENT-TEMPERATURE AND THE DNAK CHAPERONE MACHINE MODULATE THE HEAT-SHOCK RESPONSE IN ESCHERICHIA-COLI BY REGULATING THE SWITCH BETWEEN SIGMA(70) AND SIGMA(32) FACTORS ASSEMBLED WITH RNA-POLYMERASE

Escherichia coli individual sigma factors direct RNA polymerase (RNAP) to specific promoters. Upon heat shock induction there is a transient increase in the rate of transcription of similar to 20 heat shock gen es, whose promoters are recognized by the RNAP-sigma(32) rather than t he RNAP-sigma(70) holoenzyme. At least three heat shock proteins, DnaK , DnaJ and GrpE, are involved in negative modulation of the sigma(32)- dependent heat shock response. Here we show, using purified enzymes, t hat upon heat treatment of RNAP holoenzyme the sigma(70) factor is pre ferentially inactivated, whereas the resulting heat-treated RNAP core is still able to initiate transcription once supplemented with sigma(3 2) (or fresh sigma(70)). Heat-aggregated sigma(70) becomes a target fo r the joint action of DnaK, DnaJ and GrpE proteins, which reactivate i t in an ATP-dependent reaction. The RNAP-sigma(32) holoenzyme is relat ively stable at temperatures at which the RNAP-sigma(70) holoenzyme is inactivated. Furthermore, we show that formation of the RNAP-sigma(32 ) holoenzyme is favored over that of RNAP-sigma(70) at elevated temper atures. We propose a model of negative autoregulation of the heat shoc k response in which cooperative action of DnaK, DnaJ and GrpE heat sho ck proteins switches transcription back to constitutively expressed ge nes through the simultaneous reactivation of heat-aggregated sigma(70) , as well as sequestration of sigma(32) away from RNAP.