title: Yeast GCN4 transcriptional activator protein interacts with RNA polymerase II in vitro.
abstract: Regulated transcription by eukaryotic RNA polymerase II (Pol II) requires the functional interaction of multiple protein factors, some of which presumably interact directly with the polymerase. One such factor, the yeast GCN4 activator protein, binds to the upstream promoter elements of many amino acid biosynthetic genes and induces their transcription. Through the use of affinity chromatography involving GCN4- or Pol II-Sepharose columns, we show that GCN4 interacts specifically with Pol II in vitro. Purified Pol II is retained on the GCN4-Sepharose column under conditions in which the vast majority of proteins flow through. Moreover, Pol II can be selectively isolated from more complex mixtures of proteins. Conversely, GCN4 protein, synthesized in vitro or in Escherichia coli, specifically binds to the Pol II-Sepharose column under equivalent conditions. Using deletion mutants, we also show that the DNA-binding domain of GCN4 is both necessary and sufficient for this interaction. We suggest the possibility that this GCN4-Pol II interaction may be important for transcription in vivo.
authors.affiliation: Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115.
authors.forename: C J
authors.initials: CJ
authors.lastname: Brandl
authors.forename: K
authors.initials: K
authors.lastname: Struhl
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II