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. 1990 Aug;125(4):709–717. doi: 10.1093/genetics/125.4.709

Analysis of Sequence Elements Important for Expression and Regulation of the Adenylate Cyclase Gene (Cya) of Salmonella Typhimurium

L K Thorner 1, J P Fandl 1, S W Artz 1
PMCID: PMC1204097  PMID: 2204578

Abstract

We determined the nucleotide sequence of the regulatory region of the cya gene of Salmonella typhimurium. A set of nested BAL-31 deletions originating upstream of the promoter/regulatory region and extending into the cya structural gene was constructed in M13mp::cya phages and was tested for complementation of a chromosomal cya deletion mutation. BAL-31 deletion mutants unable to complement cya localized the major cya promoter. The synthetic tac promoter was inserted upstream of the BAL-31 deletions so that expression of cya was dependent on transcription from tac. Those tac derivative phages unable to complement cya localized the translation initiation region. The cya DNA sequence revealed at least three potential promoters capable of transcribing cya, with a CRP binding site straddling the -10 hexamer of the promoter proximal to the structural gene. The leader RNA sequence initiated at the latter promoter is approximately 140 bases long and includes a region that may form a stable secondary structure (ΔG = -23.8 kcal). There exist two possible in-frame translation start points, one of which is TTG and the other of which is ATG. The sequence of the S. typhimurium regulatory region was compared with that reported for Escherichia coli.

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Selected References

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

  1. Aiba H., Kawamukai M., Ishihama A. Cloning and promoter analysis of the Escherichia coli adenylate cyclase gene. Nucleic Acids Res. 1983 Jun 11;11(11):3451–3465. doi: 10.1093/nar/11.11.3451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiba H., Mori K., Tanaka M., Ooi T., Roy A., Danchin A. The complete nucleotide sequence of the adenylate cyclase gene of Escherichia coli. Nucleic Acids Res. 1984 Dec 21;12(24):9427–9440. doi: 10.1093/nar/12.24.9427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Artz S., Holzschu D., Blum P., Shand R. Use of M13mp phages to study gene regulation, structure and function: cloning and recombinational analysis of genes of the Salmonella typhimurium histidine operon. Gene. 1983 Dec;26(2-3):147–158. doi: 10.1016/0378-1119(83)90184-1. [DOI] [PubMed] [Google Scholar]
  5. Bankaitis V. A., Bassford P. J., Jr Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains. J Bacteriol. 1982 Sep;151(3):1346–1357. doi: 10.1128/jb.151.3.1346-1357.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berg O. G., von Hippel P. H. Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J Mol Biol. 1988 Apr 20;200(4):709–723. doi: 10.1016/0022-2836(88)90482-2. [DOI] [PubMed] [Google Scholar]
  7. Blum P., Blaha L., Artz S. Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium. Mol Gen Genet. 1986 Feb;202(2):327–330. doi: 10.1007/BF00331659. [DOI] [PubMed] [Google Scholar]
  8. Blum P., Holzschu D., Kwan H. S., Riggs D., Artz S. Gene replacement and retrieval with recombinant M13mp bacteriophages. J Bacteriol. 1989 Jan;171(1):538–546. doi: 10.1128/jb.171.1.538-546.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Danchin A., Guiso N., Roy A., Ullmann A. Identification of the Escherichia coli cya gene product as authentic adenylate cyclase. J Mol Biol. 1984 May 25;175(3):403–408. doi: 10.1016/0022-2836(84)90356-5. [DOI] [PubMed] [Google Scholar]
  10. Ebright R. H., Kolb A., Buc H., Kunkel T. A., Krakow J. S., Beckwith J. Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6083–6087. doi: 10.1073/pnas.84.17.6083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Irwin N., Ptashne M. Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8315–8319. doi: 10.1073/pnas.84.23.8315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jovanovich S. B. Regulation of a cya-lac fusion by cyclic AMP in Salmonella typhimurium. J Bacteriol. 1985 Feb;161(2):641–649. doi: 10.1128/jb.161.2.641-649.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kawamukai M., Kishimoto J., Utsumi R., Himeno M., Komano T., Aiba H. Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids. J Bacteriol. 1985 Nov;164(2):872–877. doi: 10.1128/jb.164.2.872-877.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koop A. H., Hartley M., Bourgeois S. Analysis of the cya locus of Escherichia coli. Gene. 1984 May;28(2):133–146. doi: 10.1016/0378-1119(84)90250-6. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leib T. K., Gerlt J. A. Evidence for a small catalytic domain in the adenylate cyclase from Salmonella typhimurium. J Biol Chem. 1983 Nov 10;258(21):12982–12987. [PubMed] [Google Scholar]
  18. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Reddy P., Peterkofsky A., McKenney K. Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5656–5660. doi: 10.1073/pnas.82.17.5656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roy A., Danchin A., Joseph E., Ullmann A. Two functional domains in adenylate cyclase of Escherichia coli. J Mol Biol. 1983 Mar 25;165(1):197–202. doi: 10.1016/s0022-2836(83)80251-4. [DOI] [PubMed] [Google Scholar]
  22. Roy A., Haziza C., Danchin A. Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region. EMBO J. 1983;2(5):791–797. doi: 10.1002/j.1460-2075.1983.tb01502.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shanblatt S. H., Revzin A. The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies. J Biol Chem. 1986 Aug 15;261(23):10885–10890. [PubMed] [Google Scholar]
  24. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  25. Straney S. B., Crothers D. M. Lac repressor is a transient gene-activating protein. Cell. 1987 Dec 4;51(5):699–707. doi: 10.1016/0092-8674(87)90093-6. [DOI] [PubMed] [Google Scholar]
  26. den Blaauwen J. L., Postma P. W. Regulation of cyclic AMP synthesis by enzyme IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system in crp strains of Salmonella typhimurium. J Bacteriol. 1985 Oct;164(1):477–478. doi: 10.1128/jb.164.1.477-478.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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