Molecular characterization of the GCN4-DNA complex.
We report studies of the DNA complex formed by GCN4, a transcriptional activator of eukaryotic amino acid biosynthetic operons. The DNA thermodynamic binding domain, defined by primer extension analysis, spans at least 18 base pairs, a site much larger than the 9-base-pair consensus defined by homology with naturally occurring binding sites. Chemical modification experiments reveal multiple sites of protein-DNA contact: methylation of any guanine N-7 or adenine N-3, ethylation of any phosphate oxygen, or elimination of any nucleoside within a region spanning nearly one and a half turns of the double helix reduces the binding affinity of the complex measurably. Nevertheless, the protein yields no detectable hydroxyl radical footprint, implying that the minor groove is reagent-accessible in the protein-DNA complex. These chemical modification patterns indicate that GCN4 does not utilize any of the DNA-recognition motifs of paradigm DNA-binding proteins. Assays to detect DNA bending induced by truncated or intact GCN4 indicate that protein conformation and not a protein-induced bend is responsible for the anomalous electrophoretic behavior of GCN4-DNA complexes.
Gartenberg
M R
MR
Department of Chemistry, Yale University, New Haven, CT 06511.
Ampe
C
C
Steitz
T A
TA
Crothers
D M
DM
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D011494:Protein Kinases