PURIFICATION AND CHARACTERIZATION OF A PROTEIN THAT BINDS TO METAL-RESPONSIVE ELEMENTS OF THE HUMAN METALLOTHIONEIN II(A) GENE

Metal responsive element (MRE) is a cis-acting DNA motif located in th e upstream region of vertebrate metallothionein genes, which can confe r metal responsiveness on downstream heterologous promoters. A protein that binds to the MRE sequence in a zinc-dependent manner (zinc regul atory factor; ZRF) was purified 16,000-fold from HeLa cell nuclear ext racts by means of the avidin-biotin method, in which a complex formed between ZRF and a biotinylated probe containing MRE was trapped by str eptavidin-agarose beads, and ZRF was recovered by salt extraction. By repeating the method three times, a homogeneous 116-kDa protein was ob tained 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. Zin c-dependent and MRE sequence-specific footprints of ZRF were obtained on MREa and MREb in the upstream region of the human metallothionein I IA, 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 homo logous to those of a mouse MRE-binding protein, mMTF-1.