Heterodimers of bHLH-PAS protein fragments derived from AhR, AhRR, and Arnt prepared by co-expression in Escherichia coli: Characterization of their DNA binding activity and preparation of a DNA complex

Yasuo Kikuchi, Shizue Ohsawa, Junsei Mimura, Masatsugu Ema, Chikahisa Takasaki, Kazuhiro Sogawa, Yoshiaki Fujii-Kuriyama

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    31 Citations (Scopus)

    Abstract

    AhR (aryl hydrocarbon receptor), AhRR (AhR repressor), and Arnt (AhR nuclear translocator) are members of the bHLH (basic-helix-loop-helix)-PAS (Per-AhR/Arnt-Sim homology sequence) transcription factor superfamily. They associate with each other to form heterodimers, AhR/Arnt or AhRR/Arnt, and bind the XRE (xenobiotic responsive element) sequences in the promoter regions of the target genes to regulate their expression. Their basic regions and HLH motifs mediate DNA binding activity and protein dimerization, respectively. The PAS domain includes two incomplete repeats, PAS-A and PAS-B, and is considered to determine the specificity on protein dimerization. However, the three-dimensional structures of PAS folds reported so far are all monomeric, therefore, little is known about the structural basis of protein interaction through PAS domains. In the present study, we prepared heterodimeric bHLH-PAS domains derived from AhR and Arnt, and AhRR and Arnt by co-expressing each pair in E. coli, and showed that the heterodimers formed exhibited full DNA-binding activity, which was not apparently affected by deletion of the highly basic amino acid cluster most N-terminal as to the HLH region of AhR or AhRR. Methylation of the two CpG sites in the XRE core sequence reduced the binding affinity to heterodimeric proteins, with 5-methylcytosine in the AhR recognition site exhibiting a greater inhibitory effect than that in the Arnt recognition site.

    Original languageEnglish
    Pages (from-to)83-90
    Number of pages8
    JournalJournal of biochemistry
    Volume134
    Issue number1
    DOIs
    Publication statusPublished - 2003 Jul 1

    Keywords

    • AhR
    • AhRR
    • Arnt
    • DNA binding
    • PAS domain

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology

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