Skip to main content
PMC 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
. 1995 Mar 28;92(7):2582–2586. doi: 10.1073/pnas.92.7.2582

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

K Fredrick 1, T Caramori 1, Y F Chen 1, A Galizzi 1, J D Helmann 1
PMCID: PMC42262  PMID: 7708689

Abstract

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Full text

PDF
2582

Images in this article

2584Fig. 3
on p.2584
2585Fig. 5
on p.2585
2585Fig. 6
on p.2585
2585Fig. 7
on p.2585

Selected References

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

  1. Adhya S., Gottesman M., Garges S., Oppenheim A. Promoter resurrection by activators--a minireview. Gene. 1993 Sep 30;132(1):1–6. doi: 10.1016/0378-1119(93)90507-y. [DOI] [PubMed] [Google Scholar]
  2. Banner C. D., Moran C. P., Jr, Losick R. Deletion analysis of a complex promoter for a developmentally regulated gene from Bacillus subtilis. J Mol Biol. 1983 Aug 5;168(2):351–365. doi: 10.1016/s0022-2836(83)80023-0. [DOI] [PubMed] [Google Scholar]
  3. Barilla D., Caramori T., Galizzi A. Coupling of flagellin gene transcription to flagellar assembly in Bacillus subtilis. J Bacteriol. 1994 Aug;176(15):4558–4564. doi: 10.1128/jb.176.15.4558-4564.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boylan S. A., Suh J. W., Thomas S. M., Price C. W. Gene encoding the alpha core subunit of Bacillus subtilis RNA polymerase is cotranscribed with the genes for initiation factor 1 and ribosomal proteins B, S13, S11, and L17. J Bacteriol. 1989 May;171(5):2553–2562. doi: 10.1128/jb.171.5.2553-2562.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bracco L., Kotlarz D., Kolb A., Diekmann S., Buc H. Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli. EMBO J. 1989 Dec 20;8(13):4289–4296. doi: 10.1002/j.1460-2075.1989.tb08615.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brunner M., Bujard H. Promoter recognition and promoter strength in the Escherichia coli system. EMBO J. 1987 Oct;6(10):3139–3144. doi: 10.1002/j.1460-2075.1987.tb02624.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen L., Helmann J. D. The Bacillus subtilis sigma D-dependent operon encoding the flagellar proteins FliD, FliS, and FliT. J Bacteriol. 1994 Jun;176(11):3093–3101. doi: 10.1128/jb.176.11.3093-3101.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ellinger T., Behnke D., Knaus R., Bujard H., Gralla J. D. Context-dependent effects of upstream A-tracts. Stimulation or inhibition of Escherichia coli promoter function. J Mol Biol. 1994 Jun 17;239(4):466–475. doi: 10.1006/jmbi.1994.1389. [DOI] [PubMed] [Google Scholar]
  9. Ferrari F. A., Trach K., Hoch J. A. Sequence analysis of the spo0B locus reveals a polycistronic transcription unit. J Bacteriol. 1985 Feb;161(2):556–562. doi: 10.1128/jb.161.2.556-562.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fredrick K. L., Helmann J. D. Dual chemotaxis signaling pathways in Bacillus subtilis: a sigma D-dependent gene encodes a novel protein with both CheW and CheY homologous domains. J Bacteriol. 1994 May;176(9):2727–2735. doi: 10.1128/jb.176.9.2727-2735.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaal T., Rao L., Estrem S. T., Yang J., Wartell R. M., Gourse R. L. Localization of the intrinsically bent DNA region upstream of the E.coli rrnB P1 promoter. Nucleic Acids Res. 1994 Jun 25;22(12):2344–2350. doi: 10.1093/nar/22.12.2344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gilman M. Z., Chamberlin M. J. Developmental and genetic regulation of Bacillus subtilis genes transcribed by sigma 28-RNA polymerase. Cell. 1983 Nov;35(1):285–293. doi: 10.1016/0092-8674(83)90231-3. [DOI] [PubMed] [Google Scholar]
  13. Graves M. C., Rabinowitz J. C. In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem. 1986 Aug 25;261(24):11409–11415. [PubMed] [Google Scholar]
  14. Helmann J. D. Alternative sigma factors and the regulation of flagellar gene expression. Mol Microbiol. 1991 Dec;5(12):2875–2882. doi: 10.1111/j.1365-2958.1991.tb01847.x. [DOI] [PubMed] [Google Scholar]
  15. Helmann J. D., Masiarz F. R., Chamberlin M. J. Isolation and characterization of the Bacillus subtilis sigma 28 factor. J Bacteriol. 1988 Apr;170(4):1560–1567. doi: 10.1128/jb.170.4.1560-1567.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Helmann J. D., Márquez L. M., Chamberlin M. J. Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene. J Bacteriol. 1988 Apr;170(4):1568–1574. doi: 10.1128/jb.170.4.1568-1574.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Juang Y. L., Helmann J. D. The delta subunit of Bacillus subtilis RNA polymerase. An allosteric effector of the initiation and core-recycling phases of transcription. J Mol Biol. 1994 May 27;239(1):1–14. doi: 10.1006/jmbi.1994.1346. [DOI] [PubMed] [Google Scholar]
  18. Knaus R., Bujard H. PL of coliphage lambda: an alternative solution for an efficient promoter. EMBO J. 1988 Sep;7(9):2919–2923. doi: 10.1002/j.1460-2075.1988.tb03150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krummel B., Chamberlin M. J. RNA chain initiation by Escherichia coli RNA polymerase. Structural transitions of the enzyme in early ternary complexes. Biochemistry. 1989 Sep 19;28(19):7829–7842. doi: 10.1021/bi00445a045. [DOI] [PubMed] [Google Scholar]
  20. Kuroda A., Sekiguchi J. High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation. J Bacteriol. 1993 Feb;175(3):795–801. doi: 10.1128/jb.175.3.795-801.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LaVallie E. R., Stahl M. L. Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation. J Bacteriol. 1989 Jun;171(6):3085–3094. doi: 10.1128/jb.171.6.3085-3094.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lampe M., Binnie C., Schmidt R., Losick R. Cloned gene encoding the delta subunit of Bacillus subtilis RNA polymerase. Gene. 1988 Jul 15;67(1):13–19. doi: 10.1016/0378-1119(88)90003-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. McAllister C. F., Achberger E. C. Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis. J Biol Chem. 1988 Aug 25;263(24):11743–11749. [PubMed] [Google Scholar]
  25. McAllister C. F., Achberger E. C. Rotational orientation of upstream curved DNA affects promoter function in Bacillus subtilis. J Biol Chem. 1989 Jun 25;264(18):10451–10456. [PubMed] [Google Scholar]
  26. Mirel D. B., Chamberlin M. J. The Bacillus subtilis flagellin gene (hag) is transcribed by the sigma 28 form of RNA polymerase. J Bacteriol. 1989 Jun;171(6):3095–3101. doi: 10.1128/jb.171.6.3095-3101.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mirel D. B., Lustre V. M., Chamberlin M. J. An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase. J Bacteriol. 1992 Jul;174(13):4197–4204. doi: 10.1128/jb.174.13.4197-4204.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  29. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Márquez L. M., Helmann J. D., Ferrari E., Parker H. M., Ordal G. W., Chamberlin M. J. Studies of sigma D-dependent functions in Bacillus subtilis. J Bacteriol. 1990 Jun;172(6):3435–3443. doi: 10.1128/jb.172.6.3435-3443.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Pérez-Martín J., Rojo F., de Lorenzo V. Promoters responsive to DNA bending: a common theme in prokaryotic gene expression. Microbiol Rev. 1994 Jun;58(2):268–290. doi: 10.1128/mr.58.2.268-290.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rao L., Ross W., Appleman J. A., Gaal T., Leirmo S., Schlax P. J., Record M. T., Jr, Gourse R. L. Factor independent activation of rrnB P1. An "extended" promoter with an upstream element that dramatically increases promoter strength. J Mol Biol. 1994 Feb 4;235(5):1421–1435. doi: 10.1006/jmbi.1994.1098. [DOI] [PubMed] [Google Scholar]
  34. Ross W., Gosink K. K., Salomon J., Igarashi K., Zou C., Ishihama A., Severinov K., Gourse R. L. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993 Nov 26;262(5138):1407–1413. doi: 10.1126/science.8248780. [DOI] [PubMed] [Google Scholar]
  35. Scotti C., Piatti M., Cuzzoni A., Perani P., Tognoni A., Grandi G., Galizzi A., Albertini A. M. A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases. Gene. 1993 Aug 16;130(1):65–71. doi: 10.1016/0378-1119(93)90347-6. [DOI] [PubMed] [Google Scholar]
  36. Shimotsu H., Henner D. J. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene. 1986;43(1-2):85–94. doi: 10.1016/0378-1119(86)90011-9. [DOI] [PubMed] [Google Scholar]
  37. Whipple F. W., Sonenshein A. L. Mechanism of initiation of transcription by Bacillus subtilis RNA polymerase at several promoters. J Mol Biol. 1992 Jan 20;223(2):399–414. doi: 10.1016/0022-2836(92)90660-c. [DOI] [PubMed] [Google Scholar]
  38. Wiggs J. L., Gilman M. Z., Chamberlin M. J. Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells. Proc Natl Acad Sci U S A. 1981 May;78(5):2762–2766. doi: 10.1073/pnas.78.5.2762. [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