Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Oct 15;90(20):9725–9729. doi: 10.1073/pnas.90.20.9725

Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

J H Alix 1, M F Guérin 1
PMCID: PMC47643  PMID: 8105482

Abstract

To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature.

Full text

PDF
9725

Selected References

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

  1. Ang D., Georgopoulos C. The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds. J Bacteriol. 1989 May;171(5):2748–2755. doi: 10.1128/jb.171.5.2748-2755.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bochner B. R., Zylicz M., Georgopoulos C. Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA. J Bacteriol. 1986 Nov;168(2):931–935. doi: 10.1128/jb.168.2.931-935.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ceccarelli A., Dotto G. P., Altruda F., Perlo C., Silengo L., Turco E., Mangiarotti G. Immature 50 S subunits in Escherichia coli polyribosomes. FEBS Lett. 1978 Sep 15;93(2):348–350. doi: 10.1016/0014-5793(78)81137-5. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Craig E. A., Gross C. A. Is hsp70 the cellular thermometer? Trends Biochem Sci. 1991 Apr;16(4):135–140. doi: 10.1016/0968-0004(91)90055-z. [DOI] [PubMed] [Google Scholar]
  6. Delaney J. M. Requirement of the Escherichia coli dnaK gene for thermotolerance and protection against H2O2. J Gen Microbiol. 1990 Oct;136(10):2113–2118. doi: 10.1099/00221287-136-10-2113. [DOI] [PubMed] [Google Scholar]
  7. Ellis R. J., van der Vies S. M. Molecular chaperones. Annu Rev Biochem. 1991;60:321–347. doi: 10.1146/annurev.bi.60.070191.001541. [DOI] [PubMed] [Google Scholar]
  8. Expert-Bezançon A., Guérin M. F., Hayes D. H., Legault L., Thibault J. Preparation of E. coli ribosomal subunits without loss of biological activity. Biochimie. 1974;56(1):77–89. doi: 10.1016/s0300-9084(74)80357-3. [DOI] [PubMed] [Google Scholar]
  9. Gaitanaris G. A., Papavassiliou A. G., Rubock P., Silverstein S. J., Gottesman M. E. Renaturation of denatured lambda repressor requires heat shock proteins. Cell. 1990 Jun 15;61(6):1013–1020. doi: 10.1016/0092-8674(90)90066-n. [DOI] [PubMed] [Google Scholar]
  10. Gaitanaris G., Papavassiliou A. G., Rubock P., Silverstein S., Gottesman M. Retraction: renaturation of denatured lambda repressor requires heat shock proteins. Cell. 1992 Sep 4;70(5):714–714. doi: 10.1016/0092-8674(92)90305-v. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Henry M. D., Yancey S. D., Kushner S. R. Role of the heat shock response in stability of mRNA in Escherichia coli K-12. J Bacteriol. 1992 Feb;174(3):743–748. doi: 10.1128/jb.174.3.743-748.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hwang D. S., Crooke E., Kornberg A. Aggregated dnaA protein is dissociated and activated for DNA replication by phospholipase or dnaK protein. J Biol Chem. 1990 Nov 5;265(31):19244–19248. [PubMed] [Google Scholar]
  14. Itikawa H., Fujita H., Wada M. High temperature induction of a stringent response in the dnaK(Ts) and dnaJ(Ts) mutants of Escherichia coli. J Biochem. 1986 Jun;99(6):1719–1724. doi: 10.1093/oxfordjournals.jbchem.a135648. [DOI] [PubMed] [Google Scholar]
  15. LaRossa R. A., Van Dyk T. K. Physiological roles of the DnaK and GroE stress proteins: catalysts of protein folding or macromolecular sponges? Mol Microbiol. 1991 Mar;5(3):529–534. doi: 10.1111/j.1365-2958.1991.tb00724.x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  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. McCarty J. S., Walker G. C. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9513–9517. doi: 10.1073/pnas.88.21.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meury J., Kohiyama M. Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli. J Bacteriol. 1991 Jul;173(14):4404–4410. doi: 10.1128/jb.173.14.4404-4410.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nelson R. J., Ziegelhoffer T., Nicolet C., Werner-Washburne M., Craig E. A. The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell. 1992 Oct 2;71(1):97–105. doi: 10.1016/0092-8674(92)90269-i. [DOI] [PubMed] [Google Scholar]
  21. Nierhaus K. H. The assembly of prokaryotic ribosomes. Biochimie. 1991 Jun;73(6):739–755. doi: 10.1016/0300-9084(91)90054-5. [DOI] [PubMed] [Google Scholar]
  22. Palleros D. R., Reid K. L., McCarty J. S., Walker G. C., Fink A. L. DnaK, hsp73, and their molten globules. Two different ways heat shock proteins respond to heat. J Biol Chem. 1992 Mar 15;267(8):5279–5285. [PubMed] [Google Scholar]
  23. Phillips G. J., Silhavy T. J. Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli. Nature. 1990 Apr 26;344(6269):882–884. doi: 10.1038/344882a0. [DOI] [PubMed] [Google Scholar]
  24. Rudd K. E., Bochner B. R., Cashel M., Roth J. R. Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression. J Bacteriol. 1985 Aug;163(2):534–542. doi: 10.1128/jb.163.2.534-542.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sherman MYu, Goldberg A. L. Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli. EMBO J. 1992 Jan;11(1):71–77. doi: 10.1002/j.1460-2075.1992.tb05029.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shi W., Zhou Y., Wild J., Adler J., Gross C. A. DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6256–6263. doi: 10.1128/jb.174.19.6256-6263.1992. [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. Spence J., Cegielska A., Georgopoulos C. Role of Escherichia coli heat shock proteins DnaK and HtpG (C62.5) in response to nutritional deprivation. J Bacteriol. 1990 Dec;172(12):7157–7166. doi: 10.1128/jb.172.12.7157-7166.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Srivastava A. K., Schlessinger D. Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7144–7148. doi: 10.1073/pnas.85.19.7144. [DOI] [PMC free article] [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. Theissen G., Behrens S. E., Wagner R. Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post-transcriptional events. Mol Microbiol. 1990 Oct;4(10):1667–1678. doi: 10.1111/j.1365-2958.1990.tb00544.x. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. VanBogelen R. A., Neidhardt F. C. Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5589–5593. doi: 10.1073/pnas.87.15.5589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wada M., Sekine K., Itikawa H. Participation of the dnaK and dnaJ gene products in phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase of Escherichia coli K-12. J Bacteriol. 1986 Oct;168(1):213–220. doi: 10.1128/jb.168.1.213-220.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Xiao H., Kalman M., Ikehara K., Zemel S., Glaser G., Cashel M. Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem. 1991 Mar 25;266(9):5980–5990. [PubMed] [Google Scholar]
  37. Zengel J. M., Lindahl L. Transcriptional control of the S10 ribosomal protein operon of Escherichia coli after a shift to higher temperature. J Bacteriol. 1985 Jul;163(1):140–147. doi: 10.1128/jb.163.1.140-147.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES