Hemiplegic mutations in AraC protein.

We have isolated mutations in AraC protein that specifically block either induction or repression at the ara pBAD promoter. These hemiplegic mutations identify amino acid residues that, correspondingly, are involved only in the induction or only in the repression activities of the protein. Residues key only for induction are 13, 15, and 18, which are located in the N-terminal arm of AraC, and residues 80 and 82 which lie in the arabinose-binding pocket of the protein's sugar-binding and dimerization domain. Alteration of residues 157, 244 and 257 can leave the protein able to activate transcription but not able to repress transcription. The behavior of the mutant proteins is consistent with the light switch mechanism for AraC action in which the presence of arabinose pulls the N-terminal arms of the protein off the DNA-binding domains, thereby freeing them to assume a direct-repeat orientation, bind to adjacent direct-repeat DNA half-sites, and activate transcription. Copyright 1999 Academic Press.