Nag repressor-operator interactions: protein-DNA contacts cover more than two turns of the DNA helix

The NagC repressor binds to two sites in the intergenic nagE-B region overlapping the divergently expressed nagE and nagB promoters. In addition the NagC repressor binds to two sites upstream of the manXYZ operon. Although basically palindromic, there is little sequence consensus between the four operators. To identify the DNA sequence important for NagC recognition, we have taken advantage of the fact that repression of the nagE and nagB genes requires the formation of a loop of DNA between molecules of the repressor bound to the nagE and nagB operators. The nagE operator was systematically mutagenised and the effect of the mutations measured on the level of expression from a nagB-lacZ fusion. These experiments showed that the most important positions for recognition are the two A.T base-pairs at positions-5 and -6 from the centre of symmetry. These are the only absolutely conserved bases in the four operators. Certain changes of residues at position -3 and -4 have fairly strong effects while changes at -7 to -10 have only minor effects. However the presence of a G or C base at positions + 11 or -11 produces a NagC binding site with considerably higher affinity than the wide-type nagE operator both in vitro and in vivo, a "super-operator". The presence of a super-operator considerably increased the stability of the binary looped NagC-DNA complex in vitro. However in the presence of cAMP/CAP, NagC showed the same apparent binding affinity to wild-type and super-operators indicating that one role of cAMP/CAP in the repression complex is to reduce the need for high affinity sites. These super-operators allow a higher level of repression of the nagE promoter compared to the nagB, presumably due to the existence of linear complexes of NagC bound to BoxE.