Purification and characterization of a protein that binds to metal responsive elements of the human metallothionein IIA gene.
Metal responsive element (MRE) is a cis-acting DNA motif located in the upstream region of vertebrate metallothionein genes, which can confer metal responsiveness on downstream heterologous promoters. A protein that binds to the MRE sequence in a zinc-dependent manner (zinc regulatory factor; ZRF) was purified 16,000-fold from HeLa cell nuclear extracts by means of the avidin-biotin method, in which a complex formed between ZRF and a biotinylated probe containing MRE was trapped by streptavidin-agarose beads, and ZRF was recovered by salt extraction. By repeating the method three times, a homogeneous 116-kDa protein was obtained whose recovery was zinc-dependent and MRE sequence-specific. UV cross-linking analysis also revealed that a protein that specifically binds to MRE has the same molecular mass as the purified protein. Zinc-dependent and MRE sequence-specific footprints of ZRF were obtained on MREa and MREb in the upstream region of the human metallothionein IIA gene. The ZRF-MRE complex dissociates by the addition of chelating reagents, suggesting a direct role of zinc ions in the DNA binding of ZRF. Partial amino acid sequences of ZRF were found to be highly homologous to those of a mouse MRE-binding protein, mMTF-1.
Otsuka
F
F
Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Iwamatsu
A
A
Suzuki
K
K
Ohsawa
M
M
Hamer
D H
DH
Koizumi
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000097809:Transcription Factor MTF-1
D004268:DNA-Binding Proteins; D008668:Metallothionein; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014157:Transcription Factors; D000097809:Transcription Factor MTF-1