Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(2):217–222. doi: 10.1002/j.1460-2075.1983.tb01408.x

Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters.

A Kolb 1, S Busby 1, M Herbert 1, D Kotlarz 1, H Buc 1
PMCID: PMC555116  PMID: 11894929

Abstract

Polyacrylamide gel electrophoresis has been used to visualise and quantitate complexes between the Escherichia coli cyclic AMP receptor protein (CRP) and DNA fragments containing the promoter region of either the E. coli galactose or lactose operons. We show that, although CRP binding to the gal fragment is weaker than binding to the lac fragment, in each case, stable complexes are formed between one dimer of CRP and one molecule of DNA. We have examined the effects of a series of deletions and point mutations in the gal promoter region on CRP binding. From the position of deletions and mutations which prevent the formation of stable complexes, we deduce the location and extent of the sequence at the CRP binding site. We show that it covers approximately the same length of sequence as the binding site at the lac promoter. Unlike the lac site, the gal site contains no palindromic sequence. We discuss the importance of symmetry in the sequence at CRP binding sites and the validity of CRP binding consensus sequences which have been proposed.

Full text

PDF
222

Images in this article

217Fig. 1.
on p.217
219Fig. 2.
on p.219

Selected References

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

  1. Bolton A. E., Hunter W. M. The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. Biochem J. 1973 Jul;133(3):529–539. doi: 10.1042/bj1330529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Busby S., Aiba H., de Crombrugghe B. Mutations in the Escherichia coli operon that define two promoters and the binding site of the cyclic AMP receptor protein. J Mol Biol. 1982 Jan 15;154(2):211–227. doi: 10.1016/0022-2836(82)90061-4. [DOI] [PubMed] [Google Scholar]
  3. Busby S., Irani M., Crombrugghe B. Isolation of mutant promoters in the Escherichia coli galactose operon using local mutagenesis on cloned DNA fragments. J Mol Biol. 1982 Jan 15;154(2):197–209. doi: 10.1016/0022-2836(82)90060-2. [DOI] [PubMed] [Google Scholar]
  4. Chapon C. Expression of malT, the regulator gene of the maltose region in Escherichia coli, is limited both at transcription and translation. EMBO J. 1982;1(3):369–374. doi: 10.1002/j.1460-2075.1982.tb01176.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dickson R. C., Abelson J., Johnson P. Nucleotide sequence changes produced by mutations in the lac promoter of Escherichia coli. J Mol Biol. 1977 Mar 25;111(1):65–75. doi: 10.1016/s0022-2836(77)80132-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Gicquel-Sanzey B., Cossart P. Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action. EMBO J. 1982;1(5):591–595. doi: 10.1002/j.1460-2075.1982.tb01213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Le Grice S. F., Matzura H., Marcoli R., Iida S., Bickle T. A. The catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene is preceded by two binding sites for the catabolite gene activator protein. J Bacteriol. 1982 Apr;150(1):312–318. doi: 10.1128/jb.150.1.312-318.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lee N. L., Gielow W. O., Wallace R. G. Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proc Natl Acad Sci U S A. 1981 Feb;78(2):752–756. doi: 10.1073/pnas.78.2.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. O'Neill M. C., Amass K., de Crombrugghe B. Molecuar model of the DNA interaction site for the cyclic AMP receptor protein. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2213–2217. doi: 10.1073/pnas.78.4.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Queen C., Rosenberg M. A promoter of pBR322 activated by cAMP receptor protein. Nucleic Acids Res. 1981 Jul 24;9(14):3365–3377. doi: 10.1093/nar/9.14.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Saxe S. A., Revzin A. Cooperative binding to DNA of catabolite activator protein of Escherichia coli. Biochemistry. 1979 Jan 23;18(2):255–263. doi: 10.1021/bi00569a003. [DOI] [PubMed] [Google Scholar]
  15. Schaeffer F., Kolb A., Buc H. Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory. EMBO J. 1982;1(1):99–105. doi: 10.1002/j.1460-2075.1982.tb01131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schmitz A. Cyclic AMP receptor proteins interacts with lactose operator DNA. Nucleic Acids Res. 1981 Jan 24;9(2):277–292. doi: 10.1093/nar/9.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Simpson R. B. Interaction of the cAMP receptor protein with the lac promoter. Nucleic Acids Res. 1980 Feb 25;8(4):759–766. [PMC free article] [PubMed] [Google Scholar]
  18. Takahashi M., Blazy B., Baudras A. Non-specific interactions of CRP from E. coli with native and denatured DNAs: control of binding by cAMP and cGMP and by cation concentration. Nucleic Acids Res. 1979 Nov 24;7(6):1699–1712. doi: 10.1093/nar/7.6.1699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Taniguchi T., O'Neill M., de Crombrugghe B. Interaction site of Escherichia coli cyclic AMP receptor protein on DNA of galactose operon promoters. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5090–5094. doi: 10.1073/pnas.76.10.5090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES