Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Feb;175(3):661–668. doi: 10.1128/jb.175.3.661-668.1993

Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro.

J T Newlands 1, T Gaal 1, J Mecsas 1, R L Gourse 1
PMCID: PMC196203  PMID: 8423142

Abstract

The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro. The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes. Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter. Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription. Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter. E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70. We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control. On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.

Full text

PDF

Images in this article

664Image
on p.664
664Image
on p.664
664Image
on p.664
664Image
on p.664
665Image
on p.665
665Image
on p.665
665Image
on p.665
666Image
on p.666

Selected References

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

  1. Borowiec J. A., Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen. EMBO J. 1988 Oct;7(10):3149–3158. doi: 10.1002/j.1460-2075.1988.tb03182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Condon C., Philips J., Fu Z. Y., Squires C., Squires C. L. Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli. EMBO J. 1992 Nov;11(11):4175–4185. doi: 10.1002/j.1460-2075.1992.tb05511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Cowing D. W., Gross C. A. Interaction of Escherichia coli RNA polymerase holoenzyme containing sigma 32 with heat shock promoters. DNase I footprinting and methylation protection. J Mol Biol. 1989 Dec 5;210(3):513–520. doi: 10.1016/0022-2836(89)90127-7. [DOI] [PubMed] [Google Scholar]
  5. Drahos D. J., Hendrix R. W. Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins. J Bacteriol. 1982 Mar;149(3):1050–1063. doi: 10.1128/jb.149.3.1050-1063.1982. [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. Gaal T., Gourse R. L. Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5533–5537. doi: 10.1073/pnas.87.14.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goff S. A., Goldberg A. L. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell. 1985 Jun;41(2):587–595. doi: 10.1016/s0092-8674(85)80031-3. [DOI] [PubMed] [Google Scholar]
  10. Gourse R. L. Visualization and quantitative analysis of complex formation between E. coli RNA polymerase and an rRNA promoter in vitro. Nucleic Acids Res. 1988 Oct 25;16(20):9789–9809. doi: 10.1093/nar/16.20.9789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gourse R. L., de Boer H. A., Nomura M. DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination. Cell. 1986 Jan 17;44(1):197–205. doi: 10.1016/0092-8674(86)90498-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Johnson W. C., Moran C. P., Jr, Losick R. Two RNA polymerase sigma factors from Bacillus subtilis discriminate between overlapping promoters for a developmentally regulated gene. Nature. 1983 Apr 28;302(5911):800–804. doi: 10.1038/302800a0. [DOI] [PubMed] [Google Scholar]
  17. Jovanovich S. B., Lesley S. A., Burgess R. R. In vitro use of monoclonal antibodies in Escherichia coli S-30 extracts to determine the RNA polymerase sigma subunit required by a promoter. J Biol Chem. 1989 Mar 5;264(7):3794–3798. [PubMed] [Google Scholar]
  18. Joyce C. M., Grindley N. D. Identification of two genes immediately downstream from the polA gene of Escherichia coli. J Bacteriol. 1982 Dec;152(3):1211–1219. doi: 10.1128/jb.152.3.1211-1219.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krueger J. H., Walker G. C. groEL and dnaK genes of Escherichia coli are induced by UV irradiation and nalidixic acid in an htpR+-dependent fashion. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1499–1503. doi: 10.1073/pnas.81.5.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kusukawa N., Yura T. Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress. Genes Dev. 1988 Jul;2(7):874–882. doi: 10.1101/gad.2.7.874. [DOI] [PubMed] [Google Scholar]
  21. Leirmo S., Gourse R. L. Factor-independent activation of Escherichia coli rRNA transcription. I. Kinetic analysis of the roles of the upstream activator region and supercoiling on transcription of the rrnB P1 promoter in vitro. J Mol Biol. 1991 Aug 5;220(3):555–568. doi: 10.1016/0022-2836(91)90100-k. [DOI] [PubMed] [Google Scholar]
  22. Leirmo S., Harrison C., Cayley D. S., Burgess R. R., Record M. T., Jr Replacement of potassium chloride by potassium glutamate dramatically enhances protein-DNA interactions in vitro. Biochemistry. 1987 Apr 21;26(8):2095–2101. doi: 10.1021/bi00382a006. [DOI] [PubMed] [Google Scholar]
  23. Lonetto M., Gribskov M., Gross C. A. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. doi: 10.1128/jb.174.12.3843-3849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Mackow E. R., Chang F. N. Correlation between RNA synthesis and ppGpp content in Escherichia coli during temperature shifts. Mol Gen Genet. 1983;192(1-2):5–9. doi: 10.1007/BF00327639. [DOI] [PubMed] [Google Scholar]
  26. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  27. Mecsas J., Cowing D. W., Gross C. A. Development of RNA polymerase-promoter contacts during open complex formation. J Mol Biol. 1991 Aug 5;220(3):585–597. doi: 10.1016/0022-2836(91)90102-c. [DOI] [PubMed] [Google Scholar]
  28. Morita M., Oka A. The structure of a transcriptional unit on colicin E1 plasmid. Eur J Biochem. 1979 Jul;97(2):435–443. doi: 10.1111/j.1432-1033.1979.tb13131.x. [DOI] [PubMed] [Google Scholar]
  29. Newlands J. T., Josaitis C. A., Ross W., Gourse R. L. Both fis-dependent and factor-independent upstream activation of the rrnB P1 promoter are face of the helix dependent. Nucleic Acids Res. 1992 Feb 25;20(4):719–726. doi: 10.1093/nar/20.4.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Newlands J. T., Ross W., Gosink K. K., Gourse R. L. Factor-independent activation of Escherichia coli rRNA transcription. II. characterization of complexes of rrnB P1 promoters containing or lacking the upstream activator region with Escherichia coli RNA polymerase. J Mol Biol. 1991 Aug 5;220(3):569–583. doi: 10.1016/0022-2836(91)90101-b. [DOI] [PubMed] [Google Scholar]
  31. Nomura M., Gourse R., Baughman G. Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem. 1984;53:75–117. doi: 10.1146/annurev.bi.53.070184.000451. [DOI] [PubMed] [Google Scholar]
  32. Peacock A. C., Dingman C. W. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968 Feb;7(2):668–674. doi: 10.1021/bi00842a023. [DOI] [PubMed] [Google Scholar]
  33. Ross W., Thompson J. F., Newlands J. T., Gourse R. L. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990 Nov;9(11):3733–3742. doi: 10.1002/j.1460-2075.1990.tb07586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ryals J., Little R., Bremer H. Control of RNA synthesis in Escherichia coli after a shift to higher temperature. J Bacteriol. 1982 Sep;151(3):1425–1432. doi: 10.1128/jb.151.3.1425-1432.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ryals J., Little R., Bremer H. Temperature dependence of RNA synthesis parameters in Escherichia coli. J Bacteriol. 1982 Aug;151(2):879–887. doi: 10.1128/jb.151.2.879-887.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sasse-Dwight S., Gralla J. D. KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J Biol Chem. 1989 May 15;264(14):8074–8081. [PubMed] [Google Scholar]
  37. Sasse-Dwight S., Gralla J. D. Probing the Escherichia coli glnALG upstream activation mechanism in vivo. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8934–8938. doi: 10.1073/pnas.85.23.8934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Tatti K. M., Moran C. P., Jr Utilization of one promoter by two forms of RNA polymerase from Bacillus subtilis. Nature. 1985 Mar 14;314(6007):190–192. doi: 10.1038/314190a0. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Tomlins R. I., Ordal Z. J. Precursor ribosomal ribonucleic acid and ribosome accumulation in vivo during the recovery of Salmonella typhimurium from thermal injury. J Bacteriol. 1971 Jul;107(1):134–142. doi: 10.1128/jb.107.1.134-142.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. VanBogelen R. A., Kelley P. M., Neidhardt F. C. Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli. J Bacteriol. 1987 Jan;169(1):26–32. doi: 10.1128/jb.169.1.26-32.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang P. Z., Doi R. H. Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem. 1984 Jul 10;259(13):8619–8625. [PubMed] [Google Scholar]
  46. Westpheling J., Ranes M., Losick R. RNA polymerase heterogeneity in Streptomyces coelicolor. Nature. 1985 Jan 3;313(5997):22–27. doi: 10.1038/313022a0. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. de Boer H., Nomura M. In vivo transcription of rRNA operons in Escherichia coli initiates with purine nucleoside triphosphates at the first promoter and with CTP at the second promoter. J Biol Chem. 1979 Jul 10;254(13):5609–5612. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES