Copper and the ACE1 regulatory protein reversibly induce yeast metallothionein gene transcription in a mouse extract

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 21; pp. 8377 - 8381
• Main Authors: Culotta, V.C. (National Institutes of Health, Bethesda, MD), Hsu, T, Hu, S, Furst, P, Hamer, D
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.11.1989; National Acad Sciences
• Subjects: Activating transcription factors; Animals; Binding sites; Biological and medical sciences; Cell Nucleus - metabolism; Cell-Free System; COBRE; COPPER; Copper - pharmacology; CUIVRE; DNA; DNA-Binding Proteins; EXTRACTOS; EXTRACTS; EXTRAIT; Fundamental and applied biological sciences. Psychology; Fungal Proteins - metabolism; GENE; Gene Expression Regulation, Fungal; General transcription factors; GENES; Genes, Fungal - drug effects; Kinetics; L Cells (Cell Line) - metabolism; LEVADURA; LEVURE; METALLOPROTEINE; METALLOPROTEINS; Metallothionein - genetics; METALPROTEINAS; Metals - pharmacology; MICE; Molecular and cellular biology; Molecular genetics; Oligonucleotides; Plasmids; Promoter Regions, Genetic; RATON; RNA; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; SOURIS; TATA box; Templates, Genetic; Transcription Factors - metabolism; Transcription, Genetic - drug effects; Transcription. Transcription factor. Splicing. Rna processing; YEASTS; In vitro transcription; Gel electrophoresis; Rodentia; DNA binding protein; Vertebrata; Mammalia; Gene; Mouse; Complexing agent; Copper I; Trans acting regulatory factor; Cyanine; Inhibition; Cell free system; Metallothionein
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.21.8377

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.