DNA sequence requirements for Ah receptor/Arnt recognition determined by in vitro transcription

The enhancer of the mouse cytochrome P450 cyp1a1 gene, which contains six binding sites (A-F) for the Ah receptor (AhR), has been a useful model system for studying the mechanism of AhR-mediated activation of transcription. In the presence of ligand, AhR interacts with its dimerization partner, Arnt, and the heteromeric complex is able to bind DNA. In the present study, we test the effects of single base pair substitutions of site D on the ability of the AhR/Arnt heteromer to recognize this response element using an in vitro transcription system. Synthetic oligodeoxyribonucleotides corresponding to the wild-type sequence of site D, or single base pair mutations of that sequence, were used to compete for AhR/Arnt binding with the transcription template. Using this competition assay, the sequence of the core recognition motif 5'-GCGTG-3' was shown to be critical for AhR/Arnt binding, and the importance of the position and orientation of the G:C and A:T base pairs of this sequence was determined.