Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding.
Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Struhl
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D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001483:Base Sequence; D005075:Biological Evolution; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016350:Leucine Zippers / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016759:Oncogene Protein p65(gag-jun) / Q000235:genetics; D011487:Protein Conformation; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D016759:Oncogene Protein p65(gag-jun); D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014498:Uracil; D001216:Asparagine; D011494:Protein Kinases; D000409:Alanine