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. 1995 Jun;177(12):3438–3442. doi: 10.1128/jb.177.12.3438-3442.1995

In vivo induction kinetics of the arabinose promoters in Escherichia coli.

C M Johnson 1, R F Schleif 1
PMCID: PMC177046  PMID: 7768852

Abstract

In Escherichia coli, the AraC protein represses transcription from its own promoter, PC, and when associated with arabinose, activates transcription from three other promoters, PBAD, PE, and PFGH. Expression from all four of these promoters is also regulated by cyclic AMP-catabolite activator protein; however, the arrangement of the protein binding sites is not identical for each promoter. We are interested in determining how the AraC protein is able to activate PBAD, PE, and PFGH despite their differences. We have characterized the induction response of the wild-type arabinose operons from their native chromosomal locations by primer extension analysis. In this analysis, mRNA from the four arabinose operons plus an internal standard could all be assayed in the RNA obtained from a single sample of cells. We found that each of the operons shows a rapid, within 15 to 30 s, response to arabinose. We also found that the expression of araFGH is more sensitive to catabolite repression but not to arabinose concentration than are araE and araBAD. Finally, we have determined the relative levels of inducibility in wild-type cells of araBAD, araFGH, and araE to be 6.5, 5, and 1, respectively. These results provide a basis for subsequent studies to determine the mechanism(s) by which AraC protein activates transcription from the different arabinose promoters.

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Selected References

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  1. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  2. Backman K., Chen Y. M., Magasanik B. Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3743–3747. doi: 10.1073/pnas.78.6.3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown C. E., Hogg R. W. A second transport system for L-arabinose in Escherichia coli B-r controlled by the araC gene. J Bacteriol. 1972 Aug;111(2):606–613. doi: 10.1128/jb.111.2.606-613.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carra J. H., Schleif R. F. Variation of half-site organization and DNA looping by AraC protein. EMBO J. 1993 Jan;12(1):35–44. doi: 10.1002/j.1460-2075.1993.tb05629.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J. Regulation of the regulatory gene for the arabinose pathway, araC. J Mol Biol. 1976 Jul 5;104(3):557–566. doi: 10.1016/0022-2836(76)90120-0. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Englesberg E., Squires C., Meronk F., Jr The L-arabinose operon in Escherichia coli B-r: a genetic demonstration of two functional states of the product of a regulator gene. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1100–1107. doi: 10.1073/pnas.62.4.1100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Greenblatt J., Schleif R. Arabinose C protein: regulation of the arabinose operon in vitro. Nat New Biol. 1971 Oct 6;233(40):166–170. doi: 10.1038/newbio233166a0. [DOI] [PubMed] [Google Scholar]
  9. Hendrickson W., Stoner C., Schleif R. Characterization of the Escherichia coli araFGH and araJ promoters. J Mol Biol. 1990 Oct 20;215(4):497–510. doi: 10.1016/S0022-2836(05)80163-9. [DOI] [PubMed] [Google Scholar]
  10. Hirsh J., Schleif R. In vivo experiments on the mechanism of action of L-arabinose C gene activator and lactose repressor. J Mol Biol. 1973 Nov 5;80(3):433–444. doi: 10.1016/0022-2836(73)90414-2. [DOI] [PubMed] [Google Scholar]
  11. Horazdovsky B. F., Hogg R. W. Genetic reconstitution of the high-affinity L-arabinose transport system. J Bacteriol. 1989 Jun;171(6):3053–3059. doi: 10.1128/jb.171.6.3053-3059.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnston R. F., Pickett S. C., Barker D. L. Autoradiography using storage phosphor technology. Electrophoresis. 1990 May;11(5):355–360. doi: 10.1002/elps.1150110503. [DOI] [PubMed] [Google Scholar]
  13. Katz L., Englesberg E. Hyperinducibility as a result of mutation in structural genes and self-catabolite repression in the ara operon. J Bacteriol. 1971 Jul;107(1):34–52. doi: 10.1128/jb.107.1.34-52.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kolodrubetz D., Schleif R. L-arabinose transport systems in Escherichia coli K-12. J Bacteriol. 1981 Nov;148(2):472–479. doi: 10.1128/jb.148.2.472-479.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kolodrubetz D., Schleif R. Regulation of the L-arabinose transport operons in Escherichia coli. J Mol Biol. 1981 Sep 15;151(2):215–227. doi: 10.1016/0022-2836(81)90512-x. [DOI] [PubMed] [Google Scholar]
  16. Kosiba B. E., Schleif R. Arabinose-inducible promoter from Escherichia coli. Its cloning from chromosomal DNA, identification as the araFG promoter and sequence. J Mol Biol. 1982 Mar 25;156(1):53–66. doi: 10.1016/0022-2836(82)90458-2. [DOI] [PubMed] [Google Scholar]
  17. Lee N., Carbon J. Nucleotide sequence of the 5' end of araBAD operon messenger RNA in Escherichia coli B/r. Proc Natl Acad Sci U S A. 1977 Jan;74(1):49–53. doi: 10.1073/pnas.74.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lis J. T., Schleif R. Different cyclic AMP requirements for induction of the arabinose and lactose operons of Escherichia coli. J Mol Biol. 1973 Sep 5;79(1):149–162. doi: 10.1016/0022-2836(73)90276-3. [DOI] [PubMed] [Google Scholar]
  19. Lobell R. B., Schleif R. F. DNA looping and unlooping by AraC protein. Science. 1990 Oct 26;250(4980):528–532. doi: 10.1126/science.2237403. [DOI] [PubMed] [Google Scholar]
  20. Lu Y., Flaherty C., Hendrickson W. AraC protein contacts asymmetric sites in the Escherichia coli araFGH promoter. J Biol Chem. 1992 Dec 5;267(34):24848–24857. [PubMed] [Google Scholar]
  21. Novotny C. P., Englesberg E. The L-arabinose permease system in Escherichia coli B/r. Biochim Biophys Acta. 1966 Mar 28;117(1):217–230. doi: 10.1016/0304-4165(66)90169-3. [DOI] [PubMed] [Google Scholar]
  22. Ogden S., Haggerty D., Stoner C. M., Kolodrubetz D., Schleif R. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3346–3350. doi: 10.1073/pnas.77.6.3346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reeder T., Schleif R. AraC protein can activate transcription from only one position and when pointed in only one direction. J Mol Biol. 1993 May 20;231(2):205–218. doi: 10.1006/jmbi.1993.1276. [DOI] [PubMed] [Google Scholar]
  24. Reeder T., Schleif R. Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon. J Bacteriol. 1991 Dec;173(24):7765–7771. doi: 10.1128/jb.173.24.7765-7771.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reichert W. L., Stein J. E., French B., Goodwin P., Varanasi U. Storage phosphor imaging technique for detection and quantitation of DNA adducts measured by the 32P-postlabeling assay. Carcinogenesis. 1992 Aug;13(8):1475–1479. doi: 10.1093/carcin/13.8.1475. [DOI] [PubMed] [Google Scholar]
  26. Stoner C. M., Schleif R. F. Transcription start site and induction kinetics of the araC regulatory gene in Escherichia coli K-12. J Mol Biol. 1983 Nov 15;170(4):1049–1053. doi: 10.1016/s0022-2836(83)80205-8. [DOI] [PubMed] [Google Scholar]
  27. Stoner C., Schleif R. The araE low affinity L-arabinose transport promoter. Cloning, sequence, transcription start site and DNA binding sites of regulatory proteins. J Mol Biol. 1983 Dec 25;171(4):369–381. doi: 10.1016/0022-2836(83)90035-9. [DOI] [PubMed] [Google Scholar]
  28. Wilcox G., Meuris P., Bass R., Englesberg E. Regulation of the L-arabinose operon BAD in vitro. J Biol Chem. 1974 May 10;249(9):2946–2952. [PubMed] [Google Scholar]

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