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.
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.
Kikuchi
Yasuo
Y
Department of Biomolecular Sciences, Graduate School of Life Science, Tohoku University, Sendai 980-8578. ykikuchi@mail.cc.tohoku.ac.jp
Ohsawa
Shizue
S
Mimura
Junsei
J
Ema
Masatsugu
M
Takasaki
Chikahisa
C
Sogawa
Kazuhiro
K
Fujii-Kuriyama
Yoshiaki
Y
D000595:Amino Acid Sequence; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D018336:Receptors, Aryl Hydrocarbon; D012097:Repressor Proteins; D014157:Transcription Factors; C492162:AHRR protein, human; C497396:ARNT protein, human