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. 1992 Aug;174(15):5005–5012. doi: 10.1128/jb.174.15.5005-5012.1992

A mutant sigma 32 with a small deletion in conserved region 3 of sigma has reduced affinity for core RNA polymerase.

Y N Zhou 1, W A Walter 1, C A Gross 1
PMCID: PMC206314  PMID: 1629156

Abstract

sigma 70, encoded by rpoD, is the major sigma factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma 70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 sigma 70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding sigma 32, an alternative sigma factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 sigma 32 continues to be made when wild-type sigma 70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of sigma 32 into the holoenzyme and free sigma suggests that RpoH113 sigma 32 has a lower binding affinity for core RNA polymerase than does wild-type sigma 32. The presence of wild-type sigma 70 exacerbates this defect. We suggest that the reduced ability of RpoH113 sigma 32 to compete with wild-type sigma 70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of sigma 32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.

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Selected References

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

  1. Baker T. A., Grossman A. D., Gross C. A. A gene regulating the heat shock response in Escherichia coli also affects proteolysis. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6779–6783. doi: 10.1073/pnas.81.21.6779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burgess R. R., Travers A. A., Dunn J. J., Bautz E. K. Factor stimulating transcription by RNA polymerase. Nature. 1969 Jan 4;221(5175):43–46. doi: 10.1038/221043a0. [DOI] [PubMed] [Google Scholar]
  3. Calendar R., Erickson J. W., Halling C., Nolte A. Deletion and insertion mutations in the rpoH gene of Escherichia coli that produce functional sigma 32. J Bacteriol. 1988 Aug;170(8):3479–3484. doi: 10.1128/jb.170.8.3479-3484.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chelm B. K., Duffy J. J., Geiduschek E. P. Interaction of Bacillus subtilis RNA polymerase core with two specificity-determining subunits. Competition between sigma and the SPO1 gene 28 protein. J Biol Chem. 1982 Jun 10;257(11):6501–6508. [PubMed] [Google Scholar]
  5. Cowing D. W., Bardwell J. C., Craig E. A., Woolford C., Hendrix R. W., Gross C. A. Consensus sequence for Escherichia coli heat shock gene promoters. Proc Natl Acad Sci U S A. 1985 May;82(9):2679–2683. doi: 10.1073/pnas.82.9.2679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Erickson J. W., Gross C. A. Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 1989 Sep;3(9):1462–1471. doi: 10.1101/gad.3.9.1462. [DOI] [PubMed] [Google Scholar]
  7. Erickson J. W., Vaughn V., Walter W. A., Neidhardt F. C., Gross C. A. Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene. Genes Dev. 1987 Jul;1(5):419–432. doi: 10.1101/gad.1.5.419. [DOI] [PubMed] [Google Scholar]
  8. Fujita N., Ishihama A. Heat-shock induction of RNA polymerase sigma-32 synthesis in Escherichia coli: transcriptional control and a multiple promoter system. Mol Gen Genet. 1987 Nov;210(1):10–15. doi: 10.1007/BF00337752. [DOI] [PubMed] [Google Scholar]
  9. Fujita N., Ishihama A., Nagasawa Y., Ueda S. RNA polymerase sigma-related proteins in Escherichia coli: detection by antibodies against a synthetic peptide. Mol Gen Genet. 1987 Nov;210(1):5–9. doi: 10.1007/BF00337751. [DOI] [PubMed] [Google Scholar]
  10. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Gross C., Hoffman J., Ward C., Hager D., Burdick G., Berger H., Burgess R. Mutation affecting thermostability of sigma subunit of Escherichia coli RNA polymerase lies near the dnaG locus at about 66 min on the E. coli genetic map. Proc Natl Acad Sci U S A. 1978 Jan;75(1):427–431. doi: 10.1073/pnas.75.1.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grossman A. D., Burgess R. R., Walter W., Gross C. A. Mutations in the Ion gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase. Cell. 1983 Jan;32(1):151–159. doi: 10.1016/0092-8674(83)90505-6. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Grossman A. D., Zhou Y. N., Gross C., Heilig J., Christie G. E., Calendar R. Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase. J Bacteriol. 1985 Mar;161(3):939–943. doi: 10.1128/jb.161.3.939-943.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harris J. D., Heilig J. S., Martinez I. I., Calendar R., Isaksson L. A. Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6177–6181. doi: 10.1073/pnas.75.12.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harris J. D., Martinez I. I., Calendar R. A gene from Escherichia coli affecting the sigma subunit of RNA polymerase. Proc Natl Acad Sci U S A. 1977 May;74(5):1836–1840. doi: 10.1073/pnas.74.5.1836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  20. Hu J. C., Gross C. A. Marker rescue with plasmids bearing deletions in rpoD identifies a dispensable part of E. coli sigma factor. Mol Gen Genet. 1983;191(3):492–498. doi: 10.1007/BF00425768. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lesley S. A., Burgess R. R. Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase. Biochemistry. 1989 Sep 19;28(19):7728–7734. doi: 10.1021/bi00445a031. [DOI] [PubMed] [Google Scholar]
  23. Lesley S. A., Thompson N. E., Burgess R. R. Studies of the role of the Escherichia coli heat shock regulatory protein sigma 32 by the use of monoclonal antibodies. J Biol Chem. 1987 Apr 15;262(11):5404–5407. [PubMed] [Google Scholar]
  24. Liebke H., Gross C., Walter W., Burgess R. A new mutation rpoD800, affecting the sigma subunit of E. coli RNA polymerase is allelic to two other sigma mutants. Mol Gen Genet. 1980 Jan;177(2):277–282. doi: 10.1007/BF00267439. [DOI] [PubMed] [Google Scholar]
  25. Losick R., Pero J. Cascades of Sigma factors. Cell. 1981 Sep;25(3):582–584. doi: 10.1016/0092-8674(81)90164-1. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Malik S., Zalenskaya K., Goldfarb A. Competition between sigma factors for core RNA polymerase. Nucleic Acids Res. 1987 Oct 26;15(20):8521–8530. doi: 10.1093/nar/15.20.8521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nagai H., Yano R., Erickson J. W., Yura T. Transcriptional regulation of the heat shock regulatory gene rpoH in Escherichia coli: involvement of a novel catabolite-sensitive promoter. J Bacteriol. 1990 May;172(5):2710–2715. doi: 10.1128/jb.172.5.2710-2715.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nakamura Y., Osawa T., Yura T. Chromosomal location of a structural gene for the RNA polymerase sigma factor in Escherichia coli. Proc Natl Acad Sci U S A. 1977 May;74(5):1831–1835. doi: 10.1073/pnas.74.5.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakamura Y. RNA polymerase mutant with altered sigma factor in Escherichia coli. Mol Gen Genet. 1978 Sep 20;165(1):1–6. doi: 10.1007/BF00270369. [DOI] [PubMed] [Google Scholar]
  31. Neidhardt F. C., VanBogelen R. A. Positive regulatory gene for temperature-controlled proteins in Escherichia coli. Biochem Biophys Res Commun. 1981 May 29;100(2):894–900. doi: 10.1016/s0006-291x(81)80257-4. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Pettijohn D. E., Clarkson K., Kossman C. R., Stonington O. G. Synthesis of ribosomal RNA on a protein-DNA complex isolated from bacteria: a comparison of ribosomal RNA synthesis in vitro and in vivo. J Mol Biol. 1970 Sep 14;52(2):281–300. doi: 10.1016/0022-2836(70)90031-8. [DOI] [PubMed] [Google Scholar]
  36. Reznikoff W. S., Siegele D. A., Cowing D. W., Gross C. A. The regulation of transcription initiation in bacteria. Annu Rev Genet. 1985;19:355–387. doi: 10.1146/annurev.ge.19.120185.002035. [DOI] [PubMed] [Google Scholar]
  37. Salser W., Gesteland R. F., Bolle A. In vitro synthesis of bacteriophage lysozyme. Nature. 1967 Aug 5;215(5101):588–591. doi: 10.1038/215588a0. [DOI] [PubMed] [Google Scholar]
  38. Skelly S., Coleman T., Fu C. F., Brot N., Weissbach H. Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8365–8369. doi: 10.1073/pnas.84.23.8365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stonington O. G., Pettijohn D. E. The folded genome of Escherichia coli isolated in a protein-DNA-RNA complex. Proc Natl Acad Sci U S A. 1971 Jan;68(1):6–9. doi: 10.1073/pnas.68.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Strickland M. S., Thompson N. E., Burgess R. R. Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. Biochemistry. 1988 Jul 26;27(15):5755–5762. doi: 10.1021/bi00415a054. [DOI] [PubMed] [Google Scholar]
  42. Taylor W. E., Straus D. B., Grossman A. D., Burton Z. F., Gross C. A., Burgess R. R. Transcription from a heat-inducible promoter causes heat shock regulation of the sigma subunit of E. coli RNA polymerase. Cell. 1984 Sep;38(2):371–381. doi: 10.1016/0092-8674(84)90492-6. [DOI] [PubMed] [Google Scholar]
  43. Williams K. P., Müller R., Rüger W., Geiduschek E. P. Overproduced bacteriophage T4 gene 33 protein binds RNA polymerase. J Bacteriol. 1989 Jun;171(6):3579–3582. doi: 10.1128/jb.171.6.3579-3582.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamamori T., Yura T. Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1982 Feb;79(3):860–864. doi: 10.1073/pnas.79.3.860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yano R., Imai M., Yura T. The use of operon fusions in studies of the heat-shock response: effects of altered sigma 32 on heat-shock promoter function in Escherichia coli. Mol Gen Genet. 1987 Apr;207(1):24–28. doi: 10.1007/BF00331486. [DOI] [PubMed] [Google Scholar]
  46. 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]

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