Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference.
C/EBP and GCN4 are basic region-leucine zipper (bZIP) DNA-binding proteins that recognize the dyad-symmetric sequences ATTGCGCAAT and ATGAGTCAT, respectively. The sequence specificities of these and other bZIP proteins are determined by their alpha-helical basic regions, which are related at the primary sequence level. To identify amino acids that are responsible for the different DNA sequence specificities of C/EBP and GCN4, two kinds of hybrid proteins were constructed: GCN4-C/EBP chimeras fused at various positions in the basic region and substitution mutants in which GCN4 basic region amino acids were replaced by the corresponding residues from C/EBP. On the basis of the DNA-binding characteristics of these hybrid proteins, three residues that contribute significantly to the differences in C/EBP and GCN4 binding specificity were defined. These residues are clustered along one face of the basic region alpha helix. Two of these specificity residues were not identified as DNA-contacting amino acids in a recently reported crystal structure of a GCN4-DNA complex, suggesting that the residues used by C/EBP and GCN4 to make base contacts are not identical. A random binding site selection procedure also was used to define the optimal recognition sequences for three of the GCN4-C/EBP fusion proteins. These experiments identify an element spanning the hinge region between the basic region and leucine zipper domains that dictates optimal half-site spacing (either directly abutted for C/EBP or overlapping by one base pair for GCN4) in high-affinity binding sites for these two proteins.
Johnson
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PF
ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016350:Leucine Zippers; D008961:Models, Structural; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011494:Protein Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D011494:Protein Kinases