Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Sep;84(17):6083–6087. doi: 10.1073/pnas.84.17.6083

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.

R H Ebright, A Kolb, H Buc, T A Kunkel, J S Krakow, J Beckwith
PMCID: PMC299012  PMID: 2888111

Abstract

It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.

Full text

PDF
6083

Selected References

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

  1. 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]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. Carter P. J., Winter G., Wilkinson A. J., Fersht A. R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell. 1984 Oct;38(3):835–840. doi: 10.1016/0092-8674(84)90278-2. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7274–7278. doi: 10.1073/pnas.81.23.7274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ebright R. H., Cossart P., Gicquel-Sanzey B., Beckwith J. Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli. Nature. 1984 Sep 20;311(5983):232–235. doi: 10.1038/311232a0. [DOI] [PubMed] [Google Scholar]
  7. Ebright R. H. Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator. Proc Natl Acad Sci U S A. 1986 Jan;83(2):303–307. doi: 10.1073/pnas.83.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ebright R. H., Le Grice S. F., Miller J. P., Krakow J. S. Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite gene activator protein (CAP) but do not stimulate binding to DNA. J Mol Biol. 1985 Mar 5;182(1):91–107. doi: 10.1016/0022-2836(85)90030-0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Fersht A. R., Shi J. P., Knill-Jones J., Lowe D. M., Wilkinson A. J., Blow D. M., Brick P., Carter P., Waye M. M., Winter G. Hydrogen bonding and biological specificity analysed by protein engineering. Nature. 1985 Mar 21;314(6008):235–238. doi: 10.1038/314235a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Gronenborn B., Messing J. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature. 1978 Mar 23;272(5651):375–377. doi: 10.1038/272375a0. [DOI] [PubMed] [Google Scholar]
  14. Jobe A., Sadler J. R., Bourgeois S. lac Repressor-operator interaction. IX. The binding of lac repressor to operators containing Oc mutations. J Mol Biol. 1974 May 15;85(2):231–248. doi: 10.1016/0022-2836(74)90362-3. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A. Mutational specificity of depurination. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1494–1498. doi: 10.1073/pnas.81.5.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kunkel T. A. The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J Biol Chem. 1985 May 10;260(9):5787–5796. [PubMed] [Google Scholar]
  18. Kunkel T. A. The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis. J Biol Chem. 1985 Oct 15;260(23):12866–12874. [PubMed] [Google Scholar]
  19. LeClerc J. E., Istock N. L., Saran B. R., Allen R., Jr Sequence analysis of ultraviolet-induced mutations in M13lacZ hybrid phage DNA. J Mol Biol. 1984 Dec 5;180(2):217–237. doi: 10.1016/s0022-2836(84)80001-7. [DOI] [PubMed] [Google Scholar]
  20. McKay D. B., Weber I. T., Steitz T. A. Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP. J Biol Chem. 1982 Aug 25;257(16):9518–9524. [PubMed] [Google Scholar]
  21. Ohlendorf D. H., Anderson W. F., Matthews B. W. Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor. J Mol Evol. 1983;19(2):109–114. doi: 10.1007/BF02300748. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Seeman N. C., Rosenberg J. M., Rich A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A. 1976 Mar;73(3):804–808. doi: 10.1073/pnas.73.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Weber I. T., McKay D. B., Steitz T. A. Two helix DNA binding motif of CAP found in lac repressor and gal repressor. Nucleic Acids Res. 1982 Aug 25;10(16):5085–5102. doi: 10.1093/nar/10.16.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weber I. T., Steitz T. A. Model of specific complex between catabolite gene activator protein and B-DNA suggested by electrostatic complementarity. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3973–3977. doi: 10.1073/pnas.81.13.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]
  27. von Hippel P. H., Berg O. G. On the specificity of DNA-protein interactions. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1608–1612. doi: 10.1073/pnas.83.6.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES