TY - JOUR
T1 - Altered DNA binding specificity of Arnt by selection of partner bHLH-PAS proteins
AU - Kinoshita, Koshi
AU - Kikuchi, Yasuo
AU - Sasakura, Yukie
AU - Suzuki, Masashi
AU - Fujii-Kuriyama, Yoshiaki
AU - Sogawa, Kazuhiro
N1 - Funding Information:
We thank Dr O. Gotoh and Dr K. Yasumoto for helpful discussions and Dr S. Adhya for the kind gift of pBEND2 vector. This work was supported in part by Grant-in-Aid for research on priority area (A) from the Ministry of Education, Science, Sports and Culture of Japan, and research for the Future Program of the Japan Society for the Promotion of Science.
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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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U2 - 10.1093/nar/gkh637
DO - 10.1093/nar/gkh637
M3 - Article
C2 - 15190133
AN - SCOPUS:3042574823
VL - 32
SP - 3169
EP - 3179
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 10
ER -