Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Oct 16;14(20):5085–5093. doi: 10.1002/j.1460-2075.1995.tb00190.x

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.

A Blaszczak 1, M Zylicz 1, C Georgopoulos 1, K Liberek 1
PMCID: PMC394611  PMID: 7588636

Abstract

In 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 approximately 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.

Full text

PDF
5085

Images in this article

5086Image
on p.5086
5087Image
on p.5087
5088Image
on p.5088
5089Image
on p.5089
5089Image
on p.5089
5090Image
on p.5090
5091Image
on p.5091

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloom M., Skelly S., VanBogelen R., Neidhardt F., Brot N., Weissbach H. In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes. J Bacteriol. 1986 May;166(2):380–384. doi: 10.1128/jb.166.2.380-384.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bukau B. Regulation of the Escherichia coli heat-shock response. Mol Microbiol. 1993 Aug;9(4):671–680. doi: 10.1111/j.1365-2958.1993.tb01727.x. [DOI] [PubMed] [Google Scholar]
  4. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  5. Cegielska A., Georgopoulos C. Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis. J Biol Chem. 1989 Dec 15;264(35):21122–21130. [PubMed] [Google Scholar]
  6. Fayet O., Louarn J. M., Georgopoulos C. Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. Mol Gen Genet. 1986 Mar;202(3):435–445. doi: 10.1007/BF00333274. [DOI] [PubMed] [Google Scholar]
  7. Gamer J., Bujard H., Bukau B. Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32. Cell. 1992 May 29;69(5):833–842. doi: 10.1016/0092-8674(92)90294-m. [DOI] [PubMed] [Google Scholar]
  8. Georgopoulos C. The emergence of the chaperone machines. Trends Biochem Sci. 1992 Aug;17(8):295–299. doi: 10.1016/0968-0004(92)90439-g. [DOI] [PubMed] [Google Scholar]
  9. Gross C. A., Grossman A. D., Liebke H., Walter W., Burgess R. R. Effects of the mutant sigma allele rpoD800 on the synthesis of specific macromolecular components of the Escherichia coli K12 cell. J Mol Biol. 1984 Jan 25;172(3):283–300. doi: 10.1016/s0022-2836(84)80027-3. [DOI] [PubMed] [Google Scholar]
  10. Grossman A. D., Erickson J. W., Gross C. A. The htpR gene product of E. coli is a sigma factor for heat-shock promoters. Cell. 1984 Sep;38(2):383–390. doi: 10.1016/0092-8674(84)90493-8. [DOI] [PubMed] [Google Scholar]
  11. Grossman A. D., Straus D. B., Walter W. A., Gross C. A. Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli. Genes Dev. 1987 Apr;1(2):179–184. doi: 10.1101/gad.1.2.179. [DOI] [PubMed] [Google Scholar]
  12. Herman C., Thévenet D., D'Ari R., Bouloc P. Degradation of sigma 32, the heat shock regulator in Escherichia coli, is governed by HflB. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3516–3520. doi: 10.1073/pnas.92.8.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Itikawa H., Ryu J. Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B. J Bacteriol. 1979 May;138(2):339–344. doi: 10.1128/jb.138.2.339-344.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  15. Liberek K., Galitski T. P., Zylicz M., Georgopoulos C. The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3516–3520. doi: 10.1073/pnas.89.8.3516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liberek K., Georgopoulos C. Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11019–11023. doi: 10.1073/pnas.90.23.11019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liberek K., Marszalek J., Ang D., Georgopoulos C., Zylicz M. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2874–2878. doi: 10.1073/pnas.88.7.2874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lowe P. A., Aebi U., Gross C., Burgess R. R. In vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpoD800) of Escherichia coli RNA polymerase proceeds by aggregation. J Biol Chem. 1981 Feb 25;256(4):2010–2015. [PubMed] [Google Scholar]
  19. Mensa-Wilmot K., Seaby R., Alfano C., Wold M. C., Gomes B., McMacken R. Reconstitution of a nine-protein system that initiates bacteriophage lambda DNA replication. J Biol Chem. 1989 Feb 15;264(5):2853–2861. [PubMed] [Google Scholar]
  20. Nagai H., Yuzawa H., Kanemori M., Yura T. A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10280–10284. doi: 10.1073/pnas.91.22.10280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neidhardt F. C., VanBogelen R. A., Vaughn V. The genetics and regulation of heat-shock proteins. Annu Rev Genet. 1984;18:295–329. doi: 10.1146/annurev.ge.18.120184.001455. [DOI] [PubMed] [Google Scholar]
  22. Osawa T., Yura T. Effects of reduced amount of RNA polymerase sigma factor on gene expression and growth of Escherichia coli: studies of the rpoD450 (amber) mutation. Mol Gen Genet. 1981;184(2):166–173. doi: 10.1007/BF00272900. [DOI] [PubMed] [Google Scholar]
  23. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  24. Raina S., Missiakas D., Georgopoulos C. The rpoE gene encoding the sigma E (sigma 24) heat shock sigma factor of Escherichia coli. EMBO J. 1995 Mar 1;14(5):1043–1055. doi: 10.1002/j.1460-2075.1995.tb07085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rouvière P. E., De Las Peñas A., Mecsas J., Lu C. Z., Rudd K. E., Gross C. A. rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli. EMBO J. 1995 Mar 1;14(5):1032–1042. doi: 10.1002/j.1460-2075.1995.tb07084.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schröder H., Langer T., Hartl F. U., Bukau B. DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 1993 Nov;12(11):4137–4144. doi: 10.1002/j.1460-2075.1993.tb06097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Skowyra D., Georgopoulos C., Zylicz M. The E. coli dnaK gene product, the hsp70 homolog, can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell. 1990 Sep 7;62(5):939–944. doi: 10.1016/0092-8674(90)90268-j. [DOI] [PubMed] [Google Scholar]
  28. Straus D. B., Walter W. A., Gross C. A. The activity of sigma 32 is reduced under conditions of excess heat shock protein production in Escherichia coli. Genes Dev. 1989 Dec;3(12A):2003–2010. doi: 10.1101/gad.3.12a.2003. [DOI] [PubMed] [Google Scholar]
  29. Straus D. B., Walter W. A., Gross C. A. The heat shock response of E. coli is regulated by changes in the concentration of sigma 32. Nature. 1987 Sep 24;329(6137):348–351. doi: 10.1038/329348a0. [DOI] [PubMed] [Google Scholar]
  30. Straus D., Walter W., Gross C. A. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev. 1990 Dec;4(12A):2202–2209. doi: 10.1101/gad.4.12a.2202. [DOI] [PubMed] [Google Scholar]
  31. Tilly K., McKittrick N., Zylicz M., Georgopoulos C. The dnaK protein modulates the heat-shock response of Escherichia coli. Cell. 1983 Sep;34(2):641–646. doi: 10.1016/0092-8674(83)90396-3. [DOI] [PubMed] [Google Scholar]
  32. Tilly K., Spence J., Georgopoulos C. Modulation of stability of the Escherichia coli heat shock regulatory factor sigma. J Bacteriol. 1989 Mar;171(3):1585–1589. doi: 10.1128/jb.171.3.1585-1589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wickner S., Hoskins J., McKenney K. Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7903–7907. doi: 10.1073/pnas.88.18.7903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yura T., Nagai H., Mori H. Regulation of the heat-shock response in bacteria. Annu Rev Microbiol. 1993;47:321–350. doi: 10.1146/annurev.mi.47.100193.001541. [DOI] [PubMed] [Google Scholar]
  35. Zhou Y. N., Gross C. A. How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32. J Bacteriol. 1992 Nov;174(22):7128–7137. doi: 10.1128/jb.174.22.7128-7137.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhou Y. N., Kusukawa N., Erickson J. W., Gross C. A., Yura T. Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor sigma 32. J Bacteriol. 1988 Aug;170(8):3640–3649. doi: 10.1128/jb.170.8.3640-3649.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ziemienowicz A., Skowyra D., Zeilstra-Ryalls J., Fayet O., Georgopoulos C., Zylicz M. Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity. J Biol Chem. 1993 Dec 5;268(34):25425–25431. [PubMed] [Google Scholar]
  38. Ziemienowicz A., Zylicz M., Floth C., Hübscher U. Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes. J Biol Chem. 1995 Jun 30;270(26):15479–15484. doi: 10.1074/jbc.270.26.15479. [DOI] [PubMed] [Google Scholar]
  39. Zubay G. The isolation and properties of CAP, the catabolite gene activator. Methods Enzymol. 1980;65(1):856–877. doi: 10.1016/s0076-6879(80)65079-4. [DOI] [PubMed] [Google Scholar]
  40. Zylicz M., Ang D., Liberek K., Georgopoulos C. Initiation of lambda DNA replication with purified host- and bacteriophage-encoded proteins: the role of the dnaK, dnaJ and grpE heat shock proteins. EMBO J. 1989 May;8(5):1601–1608. doi: 10.1002/j.1460-2075.1989.tb03544.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES