Binding of transformed Ah receptor complex to a dioxin responsive transcriptional enhancer: Evidence for two distinct heteromeric DNA-binding forms

Michael S. Denison

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

Guinea pig hepatic Ah receptor (AhR) complex was transformed in vitro to its DNA-binding form by incubation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Transformed TCDD-AhR was covalently cross-linked by U V-irradiation to a bromodeoxyuridine-substituted oligonucleotide containing its specific DNA recognition site, the dioxin responsive element (DRE). Denaturing gel electrophoresis and autoradiography identified four TCDD-inducible protein-DNA complexes, with molecular masses of approximately 97,105, and 115 kDa and a somewhat broader complex at 247 kDa. The 247-kDa complex appears to contain two distinct protein-DNA complexes of approximately 232 and 256 kDa and represents two proteins covalently cross-linked to a single DRE oligonucleotide, while the 97, 105, and 115-kDa complexes represent single protein-DRE cross-links. UV cross-linking to DRE oligonucleotides containing variable numbers of BrdU residues revealed that the 105-kDa protein, identiied as the AhR ligand-binding subunit by photoaffinity labeling with a radioiodinated AhR agonist, cross-links to the DRE core consensus (5′-GCGTG-3′); the 97- and 115-kDa non-ligand-binding proteins differentially cross-link immediately 5′-ward of the core. Overall, our results not only demonstrate that the critical protein-DNA contacts which occur between the AhR complex and the DRE are made primarily by the ligand-binding subunit but also indicate that the AhR complex exists as two distinct heteromeric DNA-binding forms, containing one 105-kDa ligand-binding subunit and either one 115- or one 97-kDa non-ligand-binding subunit.

Original languageEnglish (US)
Pages (from-to)12841-12849
Number of pages9
JournalBiochemistry®
StatePublished - 1993
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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