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.
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.
Bellorini
M
M
Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, Via Celoria 26, 20133 Milano, Italy.
Lee
D K
DK
Dantonel
J C
JC
Zemzoumi
K
K
Roeder
R G
RG
Tora
L
L
Mantovani
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins; D002499:Centrifugation, Density Gradient; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli; D006657:Histones; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D051381:Rats; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism
D023081:CCAAT-Binding Factor; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006657:Histones; C000593008:Nfyc protein, rat; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII