Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit interaction domain are specified by two separate segments.
CCAAT-binding factors A (CBF-A) and B (CBF-B) are two subunits of the heteromeric CCAAT-binding factor. Portions of CBF-A and CBF-B have a high degree of amino acid sequence identity to segments of the HAP3 and HAP2 subunits of a yeast multimeric transcription factor. We show here that the subunits of CBF interact with each other in the absence of DNA binding. This interaction was revealed by cross-linking and coimmunoprecipitation studies. Both the DNA binding and subunit interaction functions of CBF-B have been examined by mutational analysis. A segment of 83 amino acids from residues 252 to 334, which corresponds to the evolutionarily conserved portion of CBF-B, is necessary and sufficient for CBF-A-dependent DNA binding. Carboxyl-terminal deletions of this segment (or mutations in arginine residues in this carboxyl-terminal part) abolish DNA binding, but do not alter subunit interactions between CBF-A and CBF-B. Mutations in hydrophobic amino acids within the amino-terminal part of the evolutionarily conserved sequence at positions 252-334 result in loss of both DNA binding and subunit interaction activities. Our results indicate that the evolutionarily conserved segment of CBF-B contains both DNA-binding and subunit interaction domains and that the integrity of both domains is essential for DNA binding.
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Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
de Crombrugghe
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B
D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D022762:CCAAT-Enhancer-Binding Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D004247:DNA