Purification and characterization of a protein that binds to metal responsive elements of the human metallothionein IIA gene

• Published in: The Journal of biological chemistry Vol. 269; no. 38; pp. 23700 - 23707
• Main Authors: Otsuka, F, Iwamatsu, A, Suzuki, K, Ohsawa, M, Hamer, D H, Koizumi, S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 23.09.1994
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; DNA-Binding Proteins - metabolism; Gene Expression Regulation; HeLa Cells; Humans; Metallothionein - genetics; Molecular Sequence Data; Molecular Weight; Nuclear Proteins - chemistry; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; Oligonucleotide Probes - chemistry; Regulatory Sequences, Nucleic Acid; RNA, Messenger - genetics; Sequence Alignment; Sequence Homology, Amino Acid; Transcription Factor MTF-1; Transcription Factors - chemistry; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)31572-7

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.