GCN4 binds with high affinity to DNA sequences containing a single consensus half-site.
bZip proteins contain a bipartite DNA-binding motif consisting of a "leucine zipper" dimerization domain and a highly charged "basic region" that directly contacts DNA. These transcription factors form dimeric complexes with each monomer recognizing half of a symmetric or nearly symmetric DNA site. We have found that the bZip protein GCN4 can also bind with high affinity to DNA sites containing only a single GCN4 consensus half-site. Because several recent lines of evidence have suggested a role for monomeric DNA binding by bZip proteins, we investigated the structure of the GCN4.half-site complex. Quantitative DNA binding and affinity cleaving studies support a model in which GCN4 binds as a dimer, with one monomer making specific contacts to the consensus half-site and the other monomer forming nonspecific contacts that are nonetheless important for binding affinity. We also examined the folding transition induced in the basic regions of this complex upon binding DNA. Circular dichroism (CD) studies demonstrate that the basic regions of both monomers are helical, suggesting that a protein folding transition may be required for both specific and nonspecific DNA binding by GCN4.
Hollenbeck
J J
JJ
Department of Chemistry, Indiana University, Bloomington 47405, USA.
Oakley
M G
MG
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004492:Edetic Acid / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004492:Edetic Acid; D011494:Protein Kinases