Mutations in the bZIP domain of yeast GCN4 that alter DNA-binding specificity.
The bZIP class of eukaryotic transcriptional regulators utilize a distinct structural motif that consists of a leucine zipper that mediates dimerization and an adjacent basic region that directly contacts DNA. Although models of the protein-DNA complex have been proposed, the basis of DNA-binding specificity is essentially unknown. By genetically selecting for derivatives of yeast GCN4 that activate transcription from promoters containing mutant binding sites, we isolate an altered-specificity mutant in which the invariant asparagine in the basic region of bZIP proteins (Asn-235) has been changed to tryptophan. Wild-type GCN4 binds the optimal site (ATGACTCAT) with much higher affinity than the mutant site (TTGACTCAA), whereas the Trp-235 protein binds these sites with similar affinity. Moreover, the Trp-235, Ala-235, and Gln-235 derivatives differ from GCN4 in their strong discrimination against GTGACTCAC. These results suggest a direct interaction between Asn-235 and the +/- 4 position of the DNA target site and are discussed in terms of the scissors-grip and induced-fork models of bZIP proteins.
Tzamarias
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
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Struhl
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D016350:Leucine Zippers / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D011494:Protein Kinases; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D016385:TATA Box; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases