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

Biochem J. 1987 Mar 15;242(3):645-53. doi: 10.1042/bj2420645.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Chemical Precipitation
  • DNA / genetics*
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression Regulation
  • Mutation
  • Receptors, Cyclic AMP / genetics*
  • Receptors, Cyclic AMP / immunology
  • beta-Galactosidase / genetics


  • Receptors, Cyclic AMP
  • DNA
  • beta-Galactosidase