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

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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.