In the absence of cAMP· CRP, the lac promoter is quite weak because its-10 box differs significantly from the consensus sequence. A mutant lac promoter with a -10 box that has the consensus sequence does not re-quire cAMP · CRP for transcription activation. It therefore seems reasonable to propose that interactions between cAMp· CRP complex and RNA polymerase holoenzyme increase the holoenzyme's affinity for the lac promoter. Biochemical and genetic studies support this proposal.
    Thomas Steitz and coworkers determined the crystal structure for the cAMP· CRP complex bound to DNA. CRP consists of two chemically identical polypeptide chains of 209 amino acid residues. Each chain consists of an N-terminal domain and a C-terminal domain, which are connected by a hinge region containing four amino acids. The N-terminal domain consists of a series of antiparallel β-sheets that form a pocket for binding cAMP The C-terminal domain contains a helix-turn-helix motif that binds to DNA. In the absence of cAMP, CRP · DNA interactions are nonspecific and weak. However, the cAMP · CRP complex binds very tightly to a specific DNA sequence designated the activator site (AS). In the lac operon, the center of AS is 61. 5 bp upstream from the transcription start site. Many other bacterial operons are activated by cAMP · CRP (see below). Each of these operons also contains at least one AS. Comparing AS sequences reveals the following 22 bp consensus sequence with twofold symmetry.

5'-AAATGTGATCTAGATCACAT T T-3'
3'-T T TACACTAGATCTAGTGTAAA-5'

    The most highly conserved nucleotides in AS are the two TGTGA motifs. Mutations that alter nucleotides in the TGTGA motifs lead to decreased lac operon transcription. Each CRP subunit binds to half of the AS. The interactions between the C-terminal domain's helix-turn-helix motif and AS are similar to those described for Lac repressor and operator DNA. In fact, helix-turn-helix interactions with DNA are a recurring theme in DNA-protein recognition.
    Binding of cAMP· CRP to the consensus sequence is so tight that an operon containing this sequence would be permanently switched on. It is therefore not surprising that actual cAMP · CRP activator sites differ from the consensus sequence. Activator sites in different operons compete for the cAMP · CRP with the activator preferentially binding to sequences that most closely resemble the consensus sequence. cAMP· CRP bound to DNA migrates in an anomalous fashion when subjected to nondenaturing gel electrophoresis. The most likely explanation for this behavior is that the protein causes the DNA to bend when it binds to it. X-ray crystallography studies confirmed this interpretation, showing that the cAMP· CRP complex sharply bends DNA by an angle of between 80 to 90 °.
    In addition to specific contacts with AS, the cAMP · CRP complex also makes specific contacts with RNA polymerase holoenzyme. CRP mutants that have substitutions in residues 156-164 reduce or eliminate lac operon transcription activation but do not affect CRP's ability to bind or bend DNA. Thus, these residues that immediately precede the helix-turn-helix motif are essential for lac operon activation.
    Examination of cAMP · CRP structure suggests an explanation. Residues 156-164 form a β turn on the CRP surface (designated activating region 1 or AR1) that interacts with RNA polymerase holoenzyme. Additional experiments were performed to distinguish between the possibility that AR1s of both CRP subunits are required for transcription activation and the possibility that the AR1 of just one subunit (promoter proximal or promoter distal) is all that is required. CRP heterodimers were constructed in which one subunit has a wild-type DNA binding motif but a mutated AR1while the other subunit has a DNA binding motif with altered specificity but a wild-type AR1. Hybrid activator sites that had one wild-type half-site and one half-site that is only compatible with the CRP with altered DNA specificity forced the heterodimers to bind to lac promoters in two possible orientations. Transcription. Activation occurred only when wild type AR1 was present on the promoter proximal subunit.