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. 1989 Jan;8(1):325–331. doi: 10.1002/j.1460-2075.1989.tb03380.x

Purification and characterization of the deoR repressor of Escherichia coli.

L Mortensen 1, G Dandanell 1, K Hammer 1
PMCID: PMC400807  PMID: 2653814

Abstract

The deoR gene, which encodes the deor repressor protein in Escherichia coli, was fused to the strong Ptrc promoter in plasmid pKK233-2. The Ptrc promoter is kept repressed by lacI repressor to prevent cell killing. Induction of the Ptrc--deoR fusion plasmid resulted in the accumulation of 4% of the soluble protein as deoR protein. The deoR repressor protein was purified to 80% purity using conventional techniques; it has a mass of 28.5 kd and appears to exist as an octamer in solution. The deoR repressor is shown by DNase I footprinting to bind to the 16 bp palindromic sequence in the Pribnow box region of the deoP1 promoter. Also, the deoR repressor binds cooperatively in vitro to a DNA template with two deoR binding sites separated by 224 bp in keeping with the conclusion from genetic experiments that more than one operator is required for efficient repression of the deo operon.

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Selected References

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

  1. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  2. Bonekamp F., Clemmesen K., Karlström O., Jensen K. F. Mechanism of UTP-modulated attenuation at the pyrE gene of Escherichia coli: an example of operon polarity control through the coupling of translation to transcription. EMBO J. 1984 Dec 1;3(12):2857–2861. doi: 10.1002/j.1460-2075.1984.tb02220.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Buxton R. S. Fusion of the lac genes to the proximal promoters of the deo operon of Escherichia coli K12. J Gen Microbiol. 1979 Jun;112(2):241–250. doi: 10.1099/00221287-112-2-241. [DOI] [PubMed] [Google Scholar]
  4. Dandanell G., Hammer K. Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli. EMBO J. 1985 Dec 1;4(12):3333–3338. doi: 10.1002/j.1460-2075.1985.tb04085.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dandanell G., Valentin-Hansen P., Larsen J. E., Hammer K. Long-range cooperativity between gene regulatory sequences in a prokaryote. 1987 Feb 26-Mar 4Nature. 325(6107):823–826. doi: 10.1038/325823a0. [DOI] [PubMed] [Google Scholar]
  6. Dunn T. M., Hahn S., Ogden S., Schleif R. F. An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5017–5020. doi: 10.1073/pnas.81.16.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Greene P. J., Heyneker H. L., Bolivar F., Rodriguez R. L., Betlach M. C., Covarrubias A. A., Backman K., Russel D. J., Tait R., Boyer H. W. A general method for the purification of restriction enzymes. Nucleic Acids Res. 1978 Jul;5(7):2373–2380. doi: 10.1093/nar/5.7.2373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hahn S., Dunn T., Schleif R. Upstream repression and CRP stimulation of the Escherichia coli L-arabinose operon. J Mol Biol. 1984 Nov 25;180(1):61–72. doi: 10.1016/0022-2836(84)90430-3. [DOI] [PubMed] [Google Scholar]
  12. Hammer-Jespersen K., Munch-Ptersen A. Multiple regulation of nucleoside catabolizing enzymes: regulation of the deo operon by the cytR and deoR gene products. Mol Gen Genet. 1975;137(4):327–335. doi: 10.1007/BF00703258. [DOI] [PubMed] [Google Scholar]
  13. Irani M. H., Orosz L., Adhya S. A control element within a structural gene: the gal operon of Escherichia coli. Cell. 1983 Mar;32(3):783–788. doi: 10.1016/0092-8674(83)90064-8. [DOI] [PubMed] [Google Scholar]
  14. Krämer H., Niemöller M., Amouyal M., Revet B., von Wilcken-Bergmann B., Müller-Hill B. lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J. 1987 May;6(5):1481–1491. doi: 10.1002/j.1460-2075.1987.tb02390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Majumdar A., Adhya S. Demonstration of two operator elements in gal: in vitro repressor binding studies. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6100–6104. doi: 10.1073/pnas.81.19.6100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Majumdar A., Adhya S. Probing the structure of gal operator-repressor complexes. Conformation change in DNA. J Biol Chem. 1987 Sep 25;262(27):13258–13262. [PubMed] [Google Scholar]
  17. 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]
  18. Valentin-Hansen P., Aiba H., Schümperli D. The structure of tandem regulatory regions in the deo operon of Escherichia coli K12. EMBO J. 1982;1(3):317–322. doi: 10.1002/j.1460-2075.1982.tb01167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Valentin-Hansen P., Albrechtsen B., Løve Larsen J. E. DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. EMBO J. 1986 Aug;5(8):2015–2021. doi: 10.1002/j.1460-2075.1986.tb04458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Valentin-Hansen P., Højrup P., Short S. The primary structure of the DeoR repressor from Escherichia coli K-12. Nucleic Acids Res. 1985 Aug 26;13(16):5927–5936. doi: 10.1093/nar/13.16.5927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Valentin-Hansen P., Svenningsen B. A., Munch-Petersen A., Hammer-Jespersen K. Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system. Mol Gen Genet. 1978 Feb 16;159(2):191–202. doi: 10.1007/BF00270893. [DOI] [PubMed] [Google Scholar]
  22. Valentin-Hansen P. Tandem CRP binding sites in the deo operon of Escherichia coli K-12. EMBO J. 1982;1(9):1049–1054. doi: 10.1002/j.1460-2075.1982.tb01295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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