DNA-SEQUENCE REQUIREMENTS FOR AH RECEPTOR ARNT RECOGNITION DETERMINEDBY 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 m odel system for studying the mechanism of AhR-mediated activation of t ranscription. In the presence of ligand, AhR interacts with its dimeri zation partner, Arnt, and the heteromeric complex is able to bind DNA. In the present study, we test the effects of single base pair substit utions of site D on the ability of the AhR/Arnt heteromer to recognize this response element using an in vitro transcription system. Synthet ic oligodeoxyribonucleotides corresponding to the wild-type sequence o f site D, or single base pair mutations of that sequence, were used to compete for AhR/Amt binding with the transcription template. Using th is competition assay, the sequence of the core recognition motif 5'-GC GTG-3' was shown to be critical for AhR/Arnt binding, and the importan ce of the position and orientation of the G:C and A:T base pairs of th is sequence was determined.