GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA.
The eukaryotic transcriptional activator protein, GCN4, synthesized in vitro from the cloned gene, binds specifically to the promoters of yeast amino acid biosynthetic genes. Previous analysis of truncated GCN4 derivatives localized the DNA binding domain to the C-terminal 60 amino acids and revealed that the size of the GCN4 derivative and the electrophoretic mobility of the protein-DNA complex were inversely related. This observation was utilized here to develop a novel method for determining the subunit structure of DNA binding proteins. A mixture of wild-type GCN4 protein and a smaller GCN4 derivative generated three complexes with DNA, two corresponding to those observed when the proteins are present individually and one new complex of intermediate mobility. This extra complex results from the heterodimer of the two GCN4 proteins of different sizes, demonstrating that GCN4 binds DNA as a dimer. The contacts sufficient for dimerization were localized to the 60 C-terminal amino acid, DNA binding domain, suggesting that dimerization of GCN4 is a critical aspect of specific DNA binding. Furthermore, stable GCN4 dimers were formed in the absence of target DNA. These observations suggest a structural model of GCN4 protein in which a dimer binds to overlapping and non-identical half-sites, explaining why GCN4 recognition sites act bidirectionally in stimulating transcription.
Hope
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Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115.
Struhl
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D002872:Chromosome Deletion; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005796:Genes; D046911:Macromolecular Substances; D014176:Protein Biosynthesis; D011494:Protein Kinases; D029701:Saccharomyces cerevisiae Proteins; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D011494:Protein Kinases