CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues

• Published in: Nucleic acids research 1997-06, Vol.25 (11), p.2174-2181
• Main Authors: Bellorini, Marianna, Lee, Don Kun, Dantonel, Jean Christophe, Zemzoumi, Khalid, Roeder, Robert G., Tora, Laszlo, Mantovani, Roberto
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Biochemistry, Molecular Biology; CCAAT-Binding Factor - metabolism; CCAAT-Enhancer-Binding Proteins; Centrifugation, Density Gradient; DNA-Binding Proteins - metabolism; Escherichia coli; Histones; Humans; Life Sciences; Mice; Molecular Sequence Data; Molecular Weight; Protein Folding; Rats; TATA Box; TATA-Box Binding Protein; Transcription Factor TFIID; Transcription Factors - metabolism; Transcription Factors, TFII - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/25.11.2174

Both the TATA and CCAAT boxes are widespread promoter elements and their binding proteins, TBP and NF-Y, are extremely conserved in evolution. NF-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YB and NF-YC contain a putative histone-like motif, a domain also present in TBP-associated factors (TAFIIs) and in the subunits of the transcriptional repressor NC2. Immunopurification of holo-TFIID with anti-TBP and anti-TAFII100 antibodies indicates that a fraction of NF-YB associates with TFIID in the absence of NF-YA. Sedimentation velocity centrifugation experiments confirm that two pools of NF-YB, and most likely NF-YC, exist: one associated with NF-YA and binding to the CCAAT box; another involved in high molecular weight complexes. We started to dissect NF-Y-TFIID interactions by showing that: (i) NF-YB and NF-YC interact with TBP in solution, both separately and once bound to each other; (ii) short stretches of both NF-YB and NF-YC located within the evolutionary conserved domains, adjacent to the putative histone fold motifs, are necessary for TBP binding; (iii) TBP single amino acid mutants in the HS2 helix, previously shown to be defective in NC2 binding, are also unable to bind NF-YB and NF-YC.