Copper and the ACE1 regulatory protein reversibly induce yeast metallothionein gene transcription in a mouse extract.
We describe a cell-free system in which the transcription of the yeast metallothionein gene is inducible by the addition of metal ions plus a specific regulatory protein. Efficient transcription requires the complete yeast ACE1 metalloregulatory protein, including both its DNA-binding and transactivation domains; a mouse nuclear extract providing RNA polymerase and general transcription factors; a template containing the ACE1 binding site; and Cu(I). Because the binding of ACE1 to DNA is dependent on Cu, it is possible to inhibit transcription by the use of Cu-complexing agents such as CN-. We have used this specific inhibition to show that the ACE1 regulatory protein is required for the maintenance as well as the formation of a functional preinitiation complex. The ability to reversibly induce yeast metallothionein gene transcription in vitro provides a powerful system for determining the molecular mechanism of a simple eukaryotic regulatory circuit.
Culotta
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VC
Laboratory of Biochemistry, National Cancer Institute, Bethesda, MD 20892.
Hsu
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Hu
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Fürst
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P
Hamer
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D
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D003300:Copper / Q000494:pharmacology*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000187:drug effects*; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics*; D008670:Metals / Q000494:pharmacology; D051379:Mice; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
C060256:CUP2 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008670:Metals; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003300:Copper; D008668:Metallothionein