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Published online 9 June 2004

Nucleic Acids Research, 2004, Vol. 32, No. 10 3169-3179
© 2004 Oxford University Press

Altered DNA binding specificity of Arnt by selection of partner bHLH–PAS proteins

Koshi Kinoshita, Yasuo Kikuchi, Yukie Sasakura, Masashi Suzuki1, Yoshiaki Fujii-Kuriyama and Kazuhiro Sogawa*

Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980–8578, Japan and 1 AIST-NIBHT CREST Centre of Structural Biology, Higashi 1–1, Tsukuba 305–0046, Japan

*To whom correspondence should be addressed. Tel: +81 22 217 6590; Fax +81 22 217 6594; Email: sogawa{at}mail.tains.tohoku.ac.jp
Present address:
Y. Fujii-Kuriyama, Center for Tsukuba Advanced Reserch Alliance, University Tsukuba, Tsukuba, Ibaraki 305–8577, Japan

Received February 26, 2004; Revised May 3, 2004;; Accepted May 14, 2004

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.


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