Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking

The catabolite activator protein (CAP) makes no direct contact with the con sensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4) G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specif icity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of similar to 40 degrees and a twist angle of similar to 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at po sition 7 form a T:A/G:C step, which is known to be associated with DNA flex ibility. It has been proposed that specificity for T:A at position 6 is a c onsequence of formation of the DNA kink between positions 6 and 7, and of e ffects of the T:A(6)/G:C-7 step on the geometry of DNA kinking, or the ener getics of DNA kinking. In this work, we determine crystallographic structur es of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes cont aining T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this sy stem does not involve differences in the geometry of DNA kinking but, rathe r, solely differences in the energetics of DNA kinking. We further infer th at the main determinant of DNA conformation in this system is protein-DNA i nteraction, and not DNA sequence. (C) 2001 Academic Press.