A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.

Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32.

Genetic evidence indicates central roles for Hsp70 chaperones in the regulation of heat shock gene expression. This regulatory function has been postulated for Escherichia coli to rely on the direct association of DnaK (Hsp70) with the heat shock transcription factor sigma 32. This report presents evidence for the physical association of DnaK, DnaJ, and GrpE chaperones with sigma 32 in vivo. Surprisingly, an interaction of DnaJ with sigma 32 exists that is distinguishable from an interaction of DnaK and GrpE with sigma 32: addition of ATP disrupts the association of DnaK and GrpE with sigma 32, but not the association of DnaJ with sigma 32. Furthermore, DnaJ-sigma 32 and DnaK-sigma 32 associations occur independent of DnaK and DnaJ, respectively. These results suggest distinct regulatory functions of DnaJ and DnaK/GrpE.