Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking.
The bZIP DNA-binding proteins are characterized by a 50-amino-acid DNA binding and dimerization motif, consisting of a highly basic DNA-binding region ('b') followed by a leucine zipper dimerization region ('ZIP'). The best characterized bZIP DNA-binding protein is GCN4, a yeast transcriptional activator. GCN4 binds to a 9-base-pair two-fold-symmetric DNA site, 5'-A-4T-3G-2A-1C0T+1C+2A+3T+4-3' (refs 7-10). A detailed model known as the 'induced helical fork' model has been proposed for the structure of the GCN4-DNA complex. Using a site-specific bromouracil-mediated photocrosslinking method, we show here that the alanine at position 238 of GCN4 contacts, or is close to, the thymine 5-methyl of A.T at position +3 of the DNA site in the GCN4-DNA complex. Our results strongly support the induced helical fork model. Our site-specific bromouracil-mediated photocrosslinking method requires no prior information regarding the structure of the protein or the structure of the protein-DNA complex and should be generalizable to DNA-binding proteins that interact with the DNA major groove.
Blatter
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Department of Chemistry, Rutgers University, New Brunswick, New Jersey 08855.
Ebright
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Ebright
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D001976:Bromouracil / Q000378:metabolism; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D011494:Protein Kinases; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry*; D014466:Ultraviolet Rays
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D001976:Bromouracil; D011494:Protein Kinases