
Metallothioneins (MTs) constitute a class of low molecular weight, cysteine-rich, metal binding proteins which are regulated at the level of gene transcription in response to heavy metals and other adverse treatments. We have previously cloned a zinc finger factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in metallothionein promoters and shown that this factor is essential for basal and heavy metal-induced transcription. Here we report that the C-terminal part of MTF-1 downstream of the DNA binding zinc fingers harbours three different transactivation domains, namely an acidic domain, a proline-rich domain and a domain rich in serine and threonine. When fused to the heterologous DNA binding domain of the yeast factor GAL4 these activation domains function constitutively, i.e. transcription of a GAL4-driven reporter gene is not induced by heavy metals. In search of the region(s) responsible for metal induction, external and internal deletion mutations of mouse and human MTF-1 and chimeric variants thereof were tested with a reporter gene driven by a metal-responsive promoter. The N-terminal part of MTF-1 containing the zinc fingers, which are dependent on zinc for efficient DNA binding, can indeed confer a limited (3- to 4-fold) zinc-responsive transcription when fused to the heterologous activation domain of the viral VP16 protein. Another region containing the acidic and proline-rich activation domains also contributes to metal inducibility, but only in the context of intact MTF-1. This indicates that the activity of MTF-1 results from a complex interplay of different functional domains.
Metallothionein/*genetics, Threonine, Transcriptional Activation, Saccharomyces cerevisiae Proteins, Proline, Recombinant Fusion Proteins, Molecular Sequence Data, Serine/analysis, Fungal Proteins/chemistry/genetics/metabolism, Fungal Proteins, DNA/*metabolism, Serine, Humans, Promoter Regions, Genetic, Proline/analysis, Herpes Simplex Virus Protein Vmw65/chemistry/genetics, Binding Sites, Base Sequence, Zinc Fingers, Herpes Simplex Virus Protein Vmw65, DNA, Transcription Factors/*chemistry/genetics/metabolism, Hydrogen-Ion Concentration, Trans-Activation (Genetics), *Saccharomyces cerevisiae Proteins, Threonine/analysis, DNA-Binding Proteins, Metals, Mutagenesis, Hela Cells, Metallothionein, Metals/*pharmacology, Promoter Regions (Genetics), Recombinant Fusion Proteins/chemistry/metabolism, Gene Deletion, Transcription Factors, HeLa Cells, Plasmids
Metallothionein/*genetics, Threonine, Transcriptional Activation, Saccharomyces cerevisiae Proteins, Proline, Recombinant Fusion Proteins, Molecular Sequence Data, Serine/analysis, Fungal Proteins/chemistry/genetics/metabolism, Fungal Proteins, DNA/*metabolism, Serine, Humans, Promoter Regions, Genetic, Proline/analysis, Herpes Simplex Virus Protein Vmw65/chemistry/genetics, Binding Sites, Base Sequence, Zinc Fingers, Herpes Simplex Virus Protein Vmw65, DNA, Transcription Factors/*chemistry/genetics/metabolism, Hydrogen-Ion Concentration, Trans-Activation (Genetics), *Saccharomyces cerevisiae Proteins, Threonine/analysis, DNA-Binding Proteins, Metals, Mutagenesis, Hela Cells, Metallothionein, Metals/*pharmacology, Promoter Regions (Genetics), Recombinant Fusion Proteins/chemistry/metabolism, Gene Deletion, Transcription Factors, HeLa Cells, Plasmids
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