Altered DNA binding specificity of Arnt by selection of partner bHLH-PAS proteins.
The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhR-Arnt and HLF-Arnt in Escherichia coli and used them for DNA binding. The dissociation constant of HLF-Arnt-HRE was 10.4 +/- 1.6 nM. Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity. Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE. By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity.
Kinoshita
Koshi
K
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8578, Japan.
Kikuchi
Yasuo
Y
Sasakura
Yukie
Y
Suzuki
Masashi
M
Fujii-Kuriyama
Yoshiaki
Y
Sogawa
Kazuhiro
K
D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D000099162:Endothelial PAS Domain-Containing Protein 1
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D015534:Trans-Activators; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C497396:ARNT protein, human; C497397:Arnt protein, mouse