FUNCTIONAL-CHARACTERIZATION OF DNA-BINDING DOMAINS OF THE SUBUNITS OFTHE HETERODIMERIC ARYL-HYDROCARBON RECEPTOR COMPLEX IMPUTING NOVEL AND CANONICAL BASIC HELIX-LOOP-HELIX PROTEIN-DNA INTERACTIONS

The aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) belong to a novel subclass of basic helix- loop helix transcription factors, The AHR ARNT heterodimer binds to th e xenobiotic responsive element (XRE). Substitution of each of four am ino acids in the basic region of ARNT with alanine severely diminishes or abolishes XRE binding, intimating that these amino acids contact D NA bases. Three of these amino acids are conserved among basic helix-l oop-helix proteins, and the corresponding amino acids of Max and USF a re known to contact DNA bases. Alanine scanning mutagenesis of the bas ic domain of AHR and substitution with conservative amino acids at par ticular positions in this domain and in a more amino-proximal AHR segm ent previously shown to be required for XRE binding (Fukunaga, B. N., and Hankinson, O. (1996) J. Biol. Chem 271, 3743-3749) demonstrate tha t the most carboxyl-proximal amino acid position of the basic domain a nd a position within the amino-proximal segment are intolerant to amin o acid substitution with regard to XRE binding, suggesting that these two amino acids make base contacts. Amino acid positions in these AHR regions and in the ARNT basic region less adversely affected by substi tution are also identified, The amino acids at these positions may con tact the phosphodiester backbone. The apparent bipartite nature of the DNA binding region of AHR and the identity of those of its amino acid s that apparently make DNA contacts impute a novel protein-DNA binding behavior for AHR.