Orientation of the heterodimeric aryl hydrocarbon (dioxin) receptor complex on its asymmetric DNA recognition sequence.
The 2,3,7,8-tetrachlorodibenzo-p-dioxin-transformed aryl hydrocarbon receptor (AHR) complex binds to xenobiotic-responsive element (XRE) sequences in the 5' flanking region of the CYP1A1 gene, resulting in initiation of transcription. Both components of the transformed AHR complex [the ligand-binding AHR monomer and the AHR nuclear translocator (ARNT)] directly contact the XRE. These proteins belong to a novel subclass of basic helix-loop-helix transcription factors. The binding sites of AHR and ARNT on the asymmetric XRE were determined using nuclear extracts of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated Hepa-1c1c7 cells and a panel of double-stranded oligonucleotides containing XRE1 of the CYP1A1 gene (5'-TTGCGTGAGAA-3'), in which all combinations of three, two, or one of the thymines indicated were substituted by the photoreactive thymine analog 5-bromodeoxyuracil. Covalent cross-linking analysis and immunoprecipitation with antibodies specific for AHR or ARNT demonstrated that ARNT directly contacts the 3'-most thymine position, that AHR directly contacts the second thymine position, and that neither protein contacts the 5'-most thymine position. The thymine position contacted by ARNT lies within a three-nucleotide sequence (5'-GTG-3') identical to a half-site of an E-box element (5'-CACGTG-3') that is recognized by a number of other basic helix-loop-helix transcription factors. AHR binds to a portion of the XRE that does not resemble an E-box. Additional experiments demonstrated that neither protein loops over to contact residues located beyond the other's binding site.
Bacsi
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SG
Laboratory of Structural Biology and Molecular Medicine, School of Medicine, University of California, Los Angeles 90024.
Reisz-Porszasz
S
S
Hankinson
O
O
D000818:Animals; D000906:Antibodies; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D001665:Binding Sites; D001973:Bromodeoxyuridine / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004268:DNA-Binding Proteins; D005609:Free Radicals / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000378:metabolism*; D014170:Transformation, Genetic; D014498:Uracil / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism
D000906:Antibodies; C497397:Arnt protein, mouse; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005609:Free Radicals; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D015262:Xenobiotics; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D014498:Uracil; D004247:DNA; D001973:Bromodeoxyuridine