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. 1982 Feb 25;10(4):1345–1361. doi: 10.1093/nar/10.4.1345

Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein.

H Aiba, S Fujimoto, N Ozaki
PMCID: PMC320530  PMID: 6280140

Abstract

The crp gene of E. coli, which codes for cAMP receptor protein (CRP), has been cloned in the plasmid pBR322 on the basis of a genetic complementation. One of the recombinant plasmids, pHA1, was shown to direct the synthesis of CRP in a cell-free system. The location of the crp gene was determined by constructing subclones carrying various portions of pHA1. The nucleotide sequence of the crp gene has been determined. The coding region consists of 627 base pairs (bp), which specify a protein of 209 amino acids. The predicted amino acid sequence from the DNA sequence is consistent with the amino acid sequence partially known and the amino acid composition of CRP. After the coding region, there is a G-C rich inverted repeat sequence followed by a run of Ts, which could be a terminator of the crp gene. A possible promoter sequence was found about 180 bp upstream from the initiation codon and was shown to act as a promoter in vitro and in vivo. There are two dyad symmetry regions in a 167 bp leader sequence.

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Selected References

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  1. 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]
  2. Aono R., Yamasaki M., Tamura G. Changes in composition of envelope proteins in adenylate cyclase- or cyclic AMP receptor protein-deficient mutants of Escherichia coli. J Bacteriol. 1978 Nov;136(2):812–814. doi: 10.1128/jb.136.2.812-814.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Eilen E., Pampeno C., Krakow J. S. Production and properties of the alpha core derived from the cyclic adenosine monophosphate receptor protein of Escherichia coli. Biochemistry. 1978 Jun 27;17(13):2469–2473. doi: 10.1021/bi00606a001. [DOI] [PubMed] [Google Scholar]
  6. Emmer M., deCrombrugghe B., Pastan I., Perlman R. Cyclic AMP receptor protein of E. coli: its role in the synthesis of inducible enzymes. Proc Natl Acad Sci U S A. 1970 Jun;66(2):480–487. doi: 10.1073/pnas.66.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fukuda R., Taketo M., Ishihama A. Autogenous regulation of RNA polymerase beta subunit synthesis in vitro. J Biol Chem. 1978 Jul 10;253(13):4501–4504. [PubMed] [Google Scholar]
  8. Grodzicker T., Arditti R. R., Eisen H. Establishment of repression by lambdoid phage in catabolite activator protein and adenylate cyclase mutants of Escherichia coli. Proc Natl Acad Sci U S A. 1972 Feb;69(2):366–370. doi: 10.1073/pnas.69.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guidi-Rontani C., Danchin A., Ullmann A. Catabolite repression in Escherichia coli mutants lacking cyclic AMP receptor protein. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5799–5801. doi: 10.1073/pnas.77.10.5799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guiso N., Blazy B. Regulatory aspects of the cyclic AMP receptor protein in Escherichia coli K-12. Biochem Biophys Res Commun. 1980 May 14;94(1):278–283. doi: 10.1016/s0006-291x(80)80217-8. [DOI] [PubMed] [Google Scholar]
  11. Harwood C. R., Meynell E. Cyclic AMP and the production of sex pili by E. coli K-12 carrying derepressed sex factors. Nature. 1975 Apr 17;254(5501):628–660. doi: 10.1038/254628a0. [DOI] [PubMed] [Google Scholar]
  12. Hong J. S., Smith G. R., Ames B. N. Adenosine 3':5'-cyclic monophosphate concentration in the bacterial host regulates the viral decision between lysogeny and lysis. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2258–2262. doi: 10.1073/pnas.68.9.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iwakura Y., Ito K., Ishihama A. Biosynthesis of RNA polymerase in Escherichia coli. I. Control of RNA polymerase content at various growth rates. Mol Gen Genet. 1974;133(1):1–23. doi: 10.1007/BF00268673. [DOI] [PubMed] [Google Scholar]
  14. Krakow J. S., Pastan I. Cyclic adenosine monophosphate receptor: loss of cAMP-dependent DNA binding activity after proteolysis in the presence of cyclic adenosine monophosphate. Proc Natl Acad Sci U S A. 1973 Sep;70(9):2529–2533. doi: 10.1073/pnas.70.9.2529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kumar S. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli. J Bacteriol. 1976 Feb;125(2):545–555. doi: 10.1128/jb.125.2.545-555.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. MacKeen L. A., DiPeri C., Schwartz I. Reductive methylation of IF-3 and EFTu with [14C]formaldehyde and sodium cyanoborohydride. FEBS Lett. 1979 May 15;101(2):387–390. doi: 10.1016/0014-5793(79)81050-9. [DOI] [PubMed] [Google Scholar]
  19. Majerfeld I. H., Miller D., Spitz E., Rickenberg H. V. Regulation of the synthesis of adenylate cyclase in Escherichia coli by the cAMP -- cAMP receptor protein complex. Mol Gen Genet. 1981;181(4):470–475. doi: 10.1007/BF00428738. [DOI] [PubMed] [Google Scholar]
  20. Majors J. Specific binding of CAP factor to lac promoter DNA. Nature. 1975 Aug 21;256(5519):672–674. doi: 10.1038/256672a0. [DOI] [PubMed] [Google Scholar]
  21. Mallick U., Herrlich P. Regulation of synthesis of a major outer membrane protein: cyclic AMP represses Escherichia coli protein III synthesis. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5520–5523. doi: 10.1073/pnas.76.11.5520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  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. 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]
  26. 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]
  27. Pastan I., Adhya S. Cyclic adenosine 5'-monophosphate in Escherichia coli. Bacteriol Rev. 1976 Sep;40(3):527–551. doi: 10.1128/br.40.3.527-551.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. doi: 10.1073/pnas.72.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Prusiner S., Miller R. E., Valentine R. C. Adenosine 3':5'-cyclic monophosphate control of the enzymes of glutamine metabolism in Escherichia coli. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2922–2926. doi: 10.1073/pnas.69.10.2922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sauer R. T., Anderegg R. Primary structure of the lambda repressor. Biochemistry. 1978 Mar 21;17(6):1092–1100. doi: 10.1021/bi00599a024. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  34. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ullmann A., Joseph E., Danchin A. Cyclic AMP as a modulator of polarity in polycistronic transcriptional units. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3194–3197. doi: 10.1073/pnas.76.7.3194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Utsumi R., Tanabe H., Nakamoto Y., Kawamukai M., Sakai H., Himeno M., Komano T., Hirota Y. Inhibitory effect of adenosine 3',5'-phosphate on cell division of Escherichia coli K-12 mutant derivatives. J Bacteriol. 1981 Sep;147(3):1105–1109. doi: 10.1128/jb.147.3.1105-1109.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weiner A. M., Platt T., Weber K. Amino-terminal sequence analysis of proteins purified on a nanomole scale by gel electrophoresis. J Biol Chem. 1972 May 25;247(10):3242–3251. [PubMed] [Google Scholar]
  39. Wu F. Y., Nath K., Wu C. W. Conformational transitions of cyclic adenosine monophosphate receptor protein of Escherichia coli. A fluorescent probe study. Biochemistry. 1974 Jun 4;13(12):2567–2572. doi: 10.1021/bi00709a015. [DOI] [PubMed] [Google Scholar]
  40. Yamamoto T., Sobel M. E., Adams S. L., Avvedimento V. E., DiLauro R., Pastan I., de Crombrugghe B., Showalter A., Pesciotta D., Fietzek P. Construction of a recombinant bacterial plasmid containing pro-alpha 1(I) collagen DNA sequences. J Biol Chem. 1980 Mar 25;255(6):2612–2615. [PubMed] [Google Scholar]
  41. Yang R., Lis J., Wu R. Elution of DNA from agarose gels after electrophoresis. Methods Enzymol. 1979;68:176–182. doi: 10.1016/0076-6879(79)68012-6. [DOI] [PubMed] [Google Scholar]
  42. Zubay G., Schwartz D., Beckwith J. Mechanism of activation of catabolite-sensitive genes: a positive control system. Proc Natl Acad Sci U S A. 1970 May;66(1):104–110. doi: 10.1073/pnas.66.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]

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