Purification and characterization of a protein that binds to metal responsive elements of the human metallothionein IIA gene
| Autor: | Motoyasu Ohsawa, A Iwamatsu, Shinji Koizumi, Koichi Suzuki, Fuminori Otsuka, D H Hamer |
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| Rok vydání: | 1994 |
| Předmět: |
Molecular Sequence Data
Biology Regulatory Sequences Nucleic Acid Biochemistry MreB chemistry.chemical_compound Metallothionein Humans Amino Acid Sequence RNA Messenger Molecular Biology chemistry.chemical_classification Binding Sites Molecular mass Base Sequence Sequence Homology Amino Acid Nuclear Proteins Promoter Cell Biology Amino acid DNA-Binding Proteins Molecular Weight chemistry Gene Expression Regulation Biotinylation Sequence motif Oligonucleotide Probes Sequence Alignment DNA HeLa Cells Transcription Factors |
| Zdroj: | The Journal of biological chemistry. 269(38) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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