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 genes, whose promoters are recognized by the RNAP-sigma(32) rather than the 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, that upon heat treatment of RNAP holoenzyme the sigma(70) factor is preferentially inactivated, whereas the resulting heat-treated RNAP core is still able to initiate transcription once supplemented with sigma(32) (or fresh sigma(70)). Heat-aggregated sigma(70) becomes a target for the joint action of DnaK, DnaJ and GrpE proteins, which reactivate it in an ATP-dependent reaction. The RNAP-sigma(32) holoenzyme is relatively 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 temperatures. We propose a model of negative autoregulation of the heat shock response in which cooperative action of DnaK, DnaJ and GrpE heat shock proteins switches transcription back to constitutively expressed genes through the simultaneous reactivation of heat-aggregated sigma(70), as well as sequestration of sigma(32) away from RNAP.