Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking

Nature. 1992 Oct 15;359(6396):650-2. doi: 10.1038/359650a0.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Base Sequence
  • Bromouracil / metabolism
  • Cross-Linking Reagents
  • DNA-Binding Proteins / chemistry*
  • Fungal Proteins / chemistry*
  • Molecular Sequence Data
  • Peptide Fragments
  • Protein Kinases*
  • Regulatory Sequences, Nucleic Acid / genetics
  • Saccharomyces cerevisiae Proteins*
  • Transcription Factors / chemistry*
  • Ultraviolet Rays

Substances

  • Amino Acids
  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • Fungal Proteins
  • Peptide Fragments
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Bromouracil
  • Protein Kinases