Mapping the protein/DNA contact sites of the Ah receptor and Ah receptor nuclear translocator.

The Ah receptor (AHR) and its DNA binding partner, the Ah receptor nuclear translocator (ARNT), are basic helix-loop-helix proteins distinguished by their PER, AHR, ARNT, and SIM (PAS) homology regions. To identify the amino acids of the AHR.ARNT heterodimer that contact the TNGCGTG recognition sequence, we have performed deletion mapping and amino acid substitutions within the N termini of both the AHR and ARNT. The ability of the variant AHR and ARNT proteins to bind DNA and activate gene transcription was determined by the gel shift analysis and transient transfection assays. We have found that the amino acids of ARNT that contact DNA are similar to those of other basic/helix-loop-helix proteins and include glutamic acid residue 83 and arginine residues 86 and 87. Although our initial experiments indicated that DNA binding of the AHR may involve two regions that are bordered by amino acids 9-17 and amino acids 34-42, further analysis demonstrated that only amino acids 34-39 are critical for the AHR.TNGC interaction. These experiments indicate that while the structural features of the ARNT.GTG complex may closely resemble that deduced for proteins such as Max, E47, and USF, the AHR.TNGC complex may represent a unique DNA binding form of basic/helix-loop-helix proteins.