Abstract
Transcription of the
Escherichia coli melAB
operon is regulated by the MelR protein, an AraC family member whose activity is modulated by the binding of melibiose. In the absence of melibiose, MelR is unable to activate the
melAB
promoter but autoregulates its own expression by repressing the
melR
promoter. Melibiose triggers MelR-dependent activation of the
melAB
promoter and relieves MelR-dependent repression of the
melR
promoter. Twenty-nine single amino acid substitutions in MelR that result in partial melibiose-independent activation of the
melAB
promoter have been identified. Combinations of different substitutions result in almost complete melibiose-independent activation of the
melAB
promoter. MelR carrying each of the single substitutions is less able to repress the
melR
promoter, while MelR carrying some combinations of substitutions is completely unable to repress the
melR
promoter. These results argue that different conformational states of MelR are responsible for activation of the
melAB
promoter and repression of the
melR
promoter. Supporting evidence for this is provided by the isolation of substitutions in MelR that block melibiose-dependent activation of the
melAB
promoter while not changing melibiose-independent repression of the
melR
promoter. Additional experiments with a bacterial two-hybrid system suggest that interactions between MelR subunits differ according to the two conformational states.