Home LiteratureArticle Details
PMID: 3109398 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Probing the sequence-specific interaction of the cyclic AMP receptor protein with DNA by site-directed mutagenesis.

The Biochemical journal ·Vol. 242 ·No. 3 ·1987-03-15 ·Pages 645-53

Gent ME, Gronenborn AM, Davies RW, Clore GM

Abstract

Mutants in the DNA-binding helix of the cyclic AMP receptor protein (CRP), as well as mutants in a synthetic DNA-binding site derived from the sequence in the lac regulatory region, have been constructed by oligonucleotide-directed mutagenesis, and used to study the effect of selected amino acid substitutions on CRP-mediated transcriptional activity and on sequence-specific DNA binding. It has been shown that mutation of Arg-180 to Lys or Leu abolishes both CRP-mediated expression of beta-galactosidase in vivo and CRP binding of DNA as measured by immunoprecipitation. In contrast, the mutation of Arg-185 to Leu or Lys and the mutation of Lys-188 to Leu does not appear to influence these two parameters significantly. On the DNA side, both substitutions studied, namely the exchange of the G . C base pair in position 2 of the consensus T1G2T3G4A5 motif into an A . T base pair and the exchange of the A . T base pair in position 5 for a G . C base pair, abolish specific binding. Implications of these findings with respect to the present models for specific CRP-DNA recognition are discussed.

MeSH Terms
Base Sequence Binding Sites Chemical Precipitation DNA/genetics Electrophoresis, Polyacrylamide Gel Gene Expression Regulation Mutation Receptors, Cyclic AMP/genetics,immunology beta-Galactosidase/genetics
Chemicals
Receptors, Cyclic AMP DNA beta-Galactosidase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Gent M E
Gronenborn A M
Davies R W
Clore G M
References (41)
41 references, click to expand
  1. 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):9518-24 PMID: 6286624
  2. Overproduction of the cyclic AMP receptor protein of Escherichia coli and expression of the engineered C-terminal DNA-binding domain.
    Biochem J. 1986 Jun 15;236(3):643-9 PMID: 3539103
  3. How cyclic AMP and its receptor protein act in Escherichia coli.
    Cell. 1982 Jun;29(2):287-9 PMID: 6288252
  4. Catabolite gene activator protein: structure, homology with other proteins, and cyclic AMP and DNA binding.
    Cold Spring Harb Symp Quant Biol. 1983;47 Pt 1:419-26 PMID: 6305560
  5. DNA-binding proteins.
    Science. 1983 Sep 9;221(4615):1020-6 PMID: 6308768
  6. On the different binding affinities of CRP at the lac, gal and malT promoter regions.
    Nucleic Acids Res. 1983 Nov 25;11(22):7833-52 PMID: 6316274
  7. The locus of sequence-directed and protein-induced DNA bending.
    Nature. 1984 Apr 5-11;308(5959):509-13 PMID: 6323997
  8. 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 PMID: 7188180
  9. Kinked DNA in crystalline complex with EcoRI endonuclease.
    Nature. 1984 May 24-30;309(5966):327-31 PMID: 6328307
  10. Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action.
    EMBO J. 1982;1(5):591-5 PMID: 6234163
  11. Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters.
    EMBO J. 1983;2(2):217-22 PMID: 11894929
  12. Purification and DNA-binding properties of the catabolite gene activator protein.
    Proc Natl Acad Sci U S A. 1971 Jun;68(6):1222-5 PMID: 4331084
  13. 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):2529-33 PMID: 4354854
  14. Sequence-specific recognition of double helical nucleic acids by proteins.
    Proc Natl Acad Sci U S A. 1976 Mar;73(3):804-8 PMID: 1062791
  15. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  16. The alpha-helix dipole and the properties of proteins.
    Nature. 1978 Jun 8;273(5662):443-6 PMID: 661956
  17. Production and properties of the alpha core derived from the cyclic adenosine monophosphate receptor protein of Escherichia coli.
    Biochemistry. 1978 Jun 27;17(13):2469-73 PMID: 209817
  18. Construction and properties of a new cloning vehicle, allowing direct screening for recombinant plasmids.
    Mol Gen Genet. 1980;178(2):475-7 PMID: 6248731
  19. Cyclic AMP receptor proteins interacts with lactose operator DNA.
    Nucleic Acids Res. 1981 Jan 24;9(2):277-92 PMID: 6259624
  20. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA.
    Nature. 1981 Apr 30;290(5809):744-9 PMID: 6261152
  21. Structure of the cro repressor from bacteriophage lambda and its interaction with DNA.
    Nature. 1981 Apr 30;290(5809):754-8 PMID: 6452580
  22. Binding of a simian virus 40 T antigen-related protein to DNA.
    J Mol Biol. 1981 Jan 25;145(3):471-88 PMID: 6267291
  23. 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):3047-60 PMID: 6269071
  24. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis.
    Nucleic Acids Res. 1981 Dec 11;9(23):6505-25 PMID: 6275366
  25. Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein.
    Nucleic Acids Res. 1982 Feb 25;10(4):1345-61 PMID: 6280140
  26. Cloning and sequence of the crp gene of Escherichia coli K 12.
    Nucleic Acids Res. 1982 Feb 25;10(4):1363-78 PMID: 6280141
  27. Theoretical aspects of specific and non-specific equilibrium binding of proteins to DNA as studied by the nitrocellulose filter binding assay. Co-operative and non-co-operative binding to a one-dimensional lattice.
    J Mol Biol. 1982 Mar 15;155(4):447-66 PMID: 6283096
  28. The N-terminal arms of lambda repressor wrap around the operator DNA.
    Nature. 1982 Jul 29;298(5873):441-3 PMID: 7088189
  29. The operator-binding domain of lambda repressor: structure and DNA recognition.
    Nature. 1982 Jul 29;298(5873):443-7 PMID: 7088190
  30. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure.
    Nature. 1982 Jul 29;298(5873):447-51 PMID: 6896364
  31. Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins.
    Proc Natl Acad Sci U S A. 1982 May;79(10):3097-100 PMID: 6212926
  32. 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):3973-7 PMID: 6377305
  33. Protein-DNA recognition.
    Annu Rev Biochem. 1984;53:293-321 PMID: 6236744
  34. Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli.
    Nature. 1984 Sep 20-26;311(5983):232-5 PMID: 6090927
  35. Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy.
    J Mol Biol. 1984 Nov 15;179(4):751-7 PMID: 6094835
  36. 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):7274-8 PMID: 6390433
  37. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template.
    DNA. 1984 Dec;3(6):479-88 PMID: 6096101
  38. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA.
    DNA. 1985 Apr;4(2):165-70 PMID: 3996185
  39. The three-dimensional structure of trp repressor.
    Nature. 1985 Oct 31-Nov 6;317(6040):782-6 PMID: 3903514
  40. A modified two primer approach to oligonucleotide-directed in vitro mutagenesis.
    Biochimie. 1985 Jul-Aug;67(7-8):841-7 PMID: 3910112
  41. The molecular basis of DNA-protein recognition inferred from the structure of cro repressor.
    Nature. 1982 Aug 19;298(5876):718-23 PMID: 6213863
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2420645
Published
1987-03-15
Pages
645-53
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1147760
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2420645 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2