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. 1987 Feb;169(2):654–659. doi: 10.1128/jb.169.2.654-659.1987

Generation of deletions in the 3'-flanking sequences of the Escherichia coli crp gene that induce cyclic AMP suppressor functions.

J W Barton, T Melton
PMCID: PMC211828  PMID: 3027042

Abstract

The crp structural gene and its 3'-flanking sequences were subcloned into M13mp8, and in vitro deletions were constructed in both the 5' and 3' ends of the gene by using Bal 31 nuclease. Deletions ranged in size from 24 to 250 base pairs at the 5' end of crp. Sixteen deletions generated at the 3' end of the gene ranged in size from 133 to 675 base pairs. The majority of deletions extended into the crp structural gene. Another class of deletions, i.e., delta crp-4, delta crp-17, and delta crp-2, had endpoints extending in the 3'-flanking sequences external to the crp structural gene. Deletions were subcloned into pBR322 and transformed into the Escherichia coli cya crp deletion strain NCR438. Transformants containing plasmid pBM4 with the delta crp4 mutation, a deletion of 133 base pairs, were cyclic AMP independent. Strain NCR440 harboring this plasmid expressed beta-galactosidase and threonine dehydratase activities and fermented lactose, ribose, arabinose, and xylose in the absence of exogenous cyclic AMP. The delta crp-4 mutation also caused strain NCR440 to be hypersensitive to exogenous cyclic AMP. The cylic AMP receptor protein expressed in maxicells from pBM4 carrying the delta crp-4 mutation comigrated with the wild-type protein on electrophoretic gels. The delta crp-4 mutation demonstrates that sequences distal to the crp structural gene can mediate cyclic AMP suppressor functions.

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Selected References

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  1. Adhya S., Garges S. How cyclic AMP and its receptor protein act in Escherichia coli. Cell. 1982 Jun;29(2):287–289. doi: 10.1016/0092-8674(82)90145-3. [DOI] [PubMed] [Google Scholar]
  2. Aiba H. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Cell. 1983 Jan;32(1):141–149. doi: 10.1016/0092-8674(83)90504-4. [DOI] [PubMed] [Google Scholar]
  3. Aiba H., Fujimoto S., Ozaki N. Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein. Nucleic Acids Res. 1982 Feb 25;10(4):1345–1361. doi: 10.1093/nar/10.4.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aiba H., Krakow J. S. Isolation and characterization of the amino and carboxyl proximal fragments of the adenosine cyclic 3' ,5'-phosphate receptor protein of Escherichia coli. Biochemistry. 1981 Aug 4;20(16):4774–4780. doi: 10.1021/bi00519a038. [DOI] [PubMed] [Google Scholar]
  5. Alexander J. K. Suppression of defects in cyclic adenosine 3',5'-monophosphate metabolism in Escherichia coli. J Bacteriol. 1980 Oct;144(1):205–209. doi: 10.1128/jb.144.1.205-209.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Alper M. D., Ames B. N. Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants. J Bacteriol. 1978 Jan;133(1):149–157. doi: 10.1128/jb.133.1.149-157.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Botsford J. L., Drexler M. The cyclic 3',5'-adenosine monophosphate receptor protein and regulation of cyclic 3',5'-adenosine monophosphate synthesis in Escherichia coli. Mol Gen Genet. 1978 Sep 20;165(1):47–56. doi: 10.1007/BF00270375. [DOI] [PubMed] [Google Scholar]
  9. Cossart P., Gicquel-Sanzey B. Cloning and sequence of the crp gene of Escherichia coli K 12. Nucleic Acids Res. 1982 Feb 25;10(4):1363–1378. doi: 10.1093/nar/10.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cossart P., Gicquel-Sanzey B. Cloning and sequence of the crp gene of Escherichia coli K 12. Nucleic Acids Res. 1982 Feb 25;10(4):1363–1378. doi: 10.1093/nar/10.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Crombrugghe B., Chen B., Anderson W., Nissley P., Gottesman M., Pastan I., Perlman R. Lac DNA, RNA polymerase and cyclic AMP receptor protein, cyclic AMP, lac repressor and inducer are the essential elements for controlled lac transcription. Nat New Biol. 1971 Jun 2;231(22):139–142. doi: 10.1038/newbio231139a0. [DOI] [PubMed] [Google Scholar]
  12. Dessein A., Schwartz M., Ullmann A. Catabolite repression in Escherichia coli mutants lacking cyclic AMP. Mol Gen Genet. 1978 Jun 1;162(1):83–87. doi: 10.1007/BF00333853. [DOI] [PubMed] [Google Scholar]
  13. Fried M. G., Crothers D. M. Equilibrium studies of the cyclic AMP receptor protein-DNA interaction. J Mol Biol. 1984 Jan 25;172(3):241–262. doi: 10.1016/s0022-2836(84)80025-x. [DOI] [PubMed] [Google Scholar]
  14. Garges S., Adhya S. Sites of allosteric shift in the structure of the cyclic AMP receptor protein. Cell. 1985 Jul;41(3):745–751. doi: 10.1016/s0092-8674(85)80055-6. [DOI] [PubMed] [Google Scholar]
  15. George S. E., Melton T. Cloning and molecular characterization of csm mutations allowing expression of catabolite-repressible operons in the absence of exogenous cyclic AMP. J Bacteriol. 1986 May;166(2):533–540. doi: 10.1128/jb.166.2.533-540.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guarente L. P., Beckwth J., Wu A. M., Platt T. A mutation distal to the messenger RNA endpoint reduces transcription termination in the tryptophan operon in Escherichia coli. J Mol Biol. 1979 Sep 5;133(1):189–197. doi: 10.1016/0022-2836(79)90258-4. [DOI] [PubMed] [Google Scholar]
  18. Harman J. G., Dobrogosz W. J. Mechanism of CRP-mediated cya suppression in Escherichia coli. J Bacteriol. 1983 Jan;153(1):191–199. doi: 10.1128/jb.153.1.191-199.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kleckner N., Roth J., Botstein D. Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics. J Mol Biol. 1977 Oct 15;116(1):125–159. doi: 10.1016/0022-2836(77)90123-1. [DOI] [PubMed] [Google Scholar]
  20. Lee N., Wilcox G., Gielow W., Arnold J., Cleary P., Englesberg E. In vitro activation of the transcription of araBAD operon by araC activator. Proc Natl Acad Sci U S A. 1974 Mar;71(3):634–638. doi: 10.1073/pnas.71.3.634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [PubMed] [Google Scholar]
  22. Melton T., Snow L. L., Freitag C. S., Dobrogosz W. J. Isolation and characterization of cAMP suppressor mutants of Escherichia coli K12. Mol Gen Genet. 1981;182(3):480–489. doi: 10.1007/BF00293939. [DOI] [PubMed] [Google Scholar]
  23. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  24. Mitra S., Zubay G., Landy A. Evidence for the preferential binding of the catabolite gene activator protein (CAP) to DNA containing the lac promoter. Biochem Biophys Res Commun. 1975 Dec 1;67(3):857–863. doi: 10.1016/0006-291x(75)90755-x. [DOI] [PubMed] [Google Scholar]
  25. Movva R. N., Green P., Nakamura K., Inouye M. Interaction of cAMP receptor protein with the ompA gene, a gene for a major outer membrane protein of Escherichia coli. FEBS Lett. 1981 Jun 15;128(2):186–190. doi: 10.1016/0014-5793(81)80077-4. [DOI] [PubMed] [Google Scholar]
  26. Musso R. E., Di Lauro R., Adhya S., de Crombrugghe B. Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters. Cell. 1977 Nov;12(3):847–854. doi: 10.1016/0092-8674(77)90283-5. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Phillips A. T., Egan R. M., Lewis B. Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli. J Bacteriol. 1978 Sep;135(3):828–840. doi: 10.1128/jb.135.3.828-840.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Plamann M. D., Stauffer L. T., Urbanowski M. L., Stauffer G. V. Complete nucleotide sequence of the E. coli glyA gene. Nucleic Acids Res. 1983 Apr 11;11(7):2065–2075. doi: 10.1093/nar/11.7.2065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Poncz M., Solowiejczyk D., Ballantine M., Schwartz E., Surrey S. "Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4298–4302. doi: 10.1073/pnas.79.14.4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sabourin D., Beckwith J. Deletion of the Escherichia coli crp gene. J Bacteriol. 1975 Apr;122(1):338–340. doi: 10.1128/jb.122.1.338-340.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanders R., McGeoch D. A mutant transcription factor that is activated by 3':5'-cyclic guanosine monophosphate. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1017–1021. doi: 10.1073/pnas.70.4.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Singer J. T., Barbier C. S., Short S. A. Identification of the Escherichia coli deoR and cytR gene products. J Bacteriol. 1985 Sep;163(3):1095–1100. doi: 10.1128/jb.163.3.1095-1100.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]

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