Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli.
The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the sigma32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes sigma32, and the FtsH protease, which is largely responsible for sigma32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose-controlled promoters. Small increases in levels of DnaK and the DnaJ co-chaperone (< 1.5-fold of wild type) resulted in decreased level and activity of sigma32 at intermediate temperature and faster shut-off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat-denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut-off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the sigma32 levels, but the accumulated sigma32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.
Tomoyasu
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T
Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
Ogura
T
T
Tatsuta
T
T
Bukau
B
B
D049069:ATP-Dependent Proteases; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D018834:Chaperonin 60 / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004790:Enzyme Induction / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D018869:Heat-Shock Response / Q000235:genetics; D006706:Homeostasis / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D017510:Protein Folding; D012808:Sigma Factor / Q000235:genetics
D001426:Bacterial Proteins; D018834:Chaperonin 60; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D012808:Sigma Factor; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; C063842:dnaK protein, E coli