Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 May;179(10):3222–3231. doi: 10.1128/jb.179.10.3222-3231.1997

The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro.

M E Brawner 1, S G Mattern 1, M J Babcock 1, J Westpheling 1
PMCID: PMC179100  PMID: 9150217

Abstract

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Brown K. L., Wood S., Buttner M. J. Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL. Mol Microbiol. 1992 May;6(9):1133–1139. doi: 10.1111/j.1365-2958.1992.tb01551.x. [DOI] [PubMed] [Google Scholar]
  2. Buttner M J, Fearnley I M, Bibb M J. The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis. Mol Gen Genet. 1987 Aug;209(1):101–109. doi: 10.1007/BF00329843. [DOI] [PubMed] [Google Scholar]
  3. Buttner M. J., Chater K. F., Bibb M. J. Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J Bacteriol. 1990 Jun;172(6):3367–3378. doi: 10.1128/jb.172.6.3367-3378.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buttner M. J. RNA polymerase heterogeneity in Streptomyces coelicolor A3(2). Mol Microbiol. 1989 Nov;3(11):1653–1659. doi: 10.1111/j.1365-2958.1989.tb00151.x. [DOI] [PubMed] [Google Scholar]
  5. Buttner M. J., Smith A. M., Bibb M. J. At least three different RNA polymerase holoenzymes direct transcription of the agarase gene (dagA) of Streptomyces coelicolor A3(2). Cell. 1988 Feb 26;52(4):599–607. doi: 10.1016/0092-8674(88)90472-2. [DOI] [PubMed] [Google Scholar]
  6. Chater K. F., Bruton C. J., Plaskitt K. A., Buttner M. J., Méndez C., Helmann J. D. The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis. Cell. 1989 Oct 6;59(1):133–143. doi: 10.1016/0092-8674(89)90876-3. [DOI] [PubMed] [Google Scholar]
  7. Daniels D., Zuber P., Losick R. Two amino acids in an RNA polymerase sigma factor involved in the recognition of adjacent base pairs in the -10 region of a cognate promoter. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8075–8079. doi: 10.1073/pnas.87.20.8075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Decker H., Hutchinson C. R. Transcriptional analysis of the Streptomyces glaucescens tetracenomycin C biosynthesis gene cluster. J Bacteriol. 1993 Jun;175(12):3887–3892. doi: 10.1128/jb.175.12.3887-3892.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delic I., Robbins P., Westpheling J. Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1885–1889. doi: 10.1073/pnas.89.5.1885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fornwald J. A., Schmidt F. J., Adams C. W., Rosenberg M., Brawner M. E. Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2130–2134. doi: 10.1073/pnas.84.8.2130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Forsman M., Granström M. Mutagenic analysis of the promoter of the Streptomyces fradiae beta-lactamase-encoding gene. Gene. 1992 Nov 2;121(1):87–94. doi: 10.1016/0378-1119(92)90165-l. [DOI] [PubMed] [Google Scholar]
  12. Gardella T., Moyle H., Susskind M. M. A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity. J Mol Biol. 1989 Apr 20;206(4):579–590. doi: 10.1016/0022-2836(89)90567-6. [DOI] [PubMed] [Google Scholar]
  13. Gralla J. D. Transcriptional control--lessons from an E. coli promoter data base. Cell. 1991 Aug 9;66(3):415–418. doi: 10.1016/0092-8674(81)90001-5. [DOI] [PubMed] [Google Scholar]
  14. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  15. Haldenwang W. G., Losick R. Novel RNA polymerase sigma factor from Bacillus subtilis. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7000–7004. doi: 10.1073/pnas.77.12.7000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hintz W. E., Lagosky P. A. A glucose-derepressed promoter for expression of heterologous products in the filamentous fungus Aspergillus nidulans. Biotechnology (N Y) 1993 Jul;11(7):815–818. doi: 10.1038/nbt0793-815. [DOI] [PubMed] [Google Scholar]
  17. Hoffmann A., Roeder R. G. Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res. 1991 Nov 25;19(22):6337–6338. doi: 10.1093/nar/19.22.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ingram C., Brawner M., Youngman P., Westpheling J. xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter. J Bacteriol. 1989 Dec;171(12):6617–6624. doi: 10.1128/jb.171.12.6617-6624.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Janssen G. R., Ward J. M., Bibb M. J. Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae. Genes Dev. 1989 Mar;3(3):415–429. doi: 10.1101/gad.3.3.415. [DOI] [PubMed] [Google Scholar]
  20. Kenney T. J., York K., Youngman P., Moran C. P., Jr Genetic evidence that RNA polymerase associated with sigma A factor uses a sporulation-specific promoter in Bacillus subtilis. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9109–9113. doi: 10.1073/pnas.86.23.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lonetto M. A., Brown K. L., Rudd K. E., Buttner M. J. Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7573–7577. doi: 10.1073/pnas.91.16.7573. [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. Mandecki W., Reznikoff W. S. A lac promoter with a changed distance between -10 and -35 regions. Nucleic Acids Res. 1982 Feb 11;10(3):903–912. doi: 10.1093/nar/10.3.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mattern S. G., Brawner M. E., Westpheling J. Identification of a complex operator for galP1, the glucose-sensitive, galactose-dependent promoter of the Streptomyces galactose operon. J Bacteriol. 1993 Mar;175(5):1213–1220. doi: 10.1128/jb.175.5.1213-1220.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  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., Brosius J., McClure W. R. Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter. J Biol Chem. 1985 Mar 25;260(6):3529–3538. [PubMed] [Google Scholar]
  30. Potúcková L., Kelemen G. H., Findlay K. C., Lonetto M. A., Buttner M. J., Kormanec J. A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp. Mol Microbiol. 1995 Jul;17(1):37–48. doi: 10.1111/j.1365-2958.1995.mmi_17010037.x. [DOI] [PubMed] [Google Scholar]
  31. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Siegele D. A., Hu J. C., Walter W. A., Gross C. A. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol. 1989 Apr 20;206(4):591–603. doi: 10.1016/0022-2836(89)90568-8. [DOI] [PubMed] [Google Scholar]
  34. Smith C. P., Chater K. F. Structure and regulation of controlling sequences for the Streptomyces coelicolor glycerol operon. J Mol Biol. 1988 Dec 5;204(3):569–580. doi: 10.1016/0022-2836(88)90356-7. [DOI] [PubMed] [Google Scholar]
  35. Stefano J. E., Gralla J. D. Spacer mutations in the lac ps promoter. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1069–1072. doi: 10.1073/pnas.79.4.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Strohl W. R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992 Mar 11;20(5):961–974. doi: 10.1093/nar/20.5.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tanaka K., Shiina T., Takahashi H. Multiple principal sigma factor homologs in eubacteria: identification of the "rpoD box". Science. 1988 Nov 18;242(4881):1040–1042. doi: 10.1126/science.3194753. [DOI] [PubMed] [Google Scholar]
  38. Tatti K. M., Jones C. H., Moran C. P., Jr Genetic evidence for interaction of sigma E with the spoIIID promoter in Bacillus subtilis. J Bacteriol. 1991 Dec;173(24):7828–7833. doi: 10.1128/jb.173.24.7828-7833.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Westpheling J., Brawner M. Two transcribing activities are involved in expression of the Streptomyces galactose operon. J Bacteriol. 1989 Mar;171(3):1355–1361. doi: 10.1128/jb.171.3.1355-1361.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Youderian P., Bouvier S., Susskind M. M. Sequence determinants of promoter activity. Cell. 1982 Oct;30(3):843–853. doi: 10.1016/0092-8674(82)90289-6. [DOI] [PubMed] [Google Scholar]
  42. Zuber P., Healy J., Carter H. L., 3rd, Cutting S., Moran C. P., Jr, Losick R. Mutation changing the specificity of an RNA polymerase sigma factor. J Mol Biol. 1989 Apr 20;206(4):605–614. doi: 10.1016/0022-2836(89)90569-x. [DOI] [PubMed] [Google Scholar]
  43. Zukowski M. M., Gaffney D. F., Speck D., Kauffmann M., Findeli A., Wisecup A., Lecocq J. P. Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1101–1105. doi: 10.1073/pnas.80.4.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES