NF-Y histone fold alpha1 helices help impart CCAAT specificity.
NF-Y is a conserved trimeric transcriptional activator with an extremely high specificity for CCAAT boxes. The NF-YB and NF-YC subunits have histone fold motifs with a high degree of homology to NC2alpha/beta, a TBP-binding repressor. The histone fold is composed of three alpha helices, alpha1, alpha2, alpha3, separated by short loops. Structural data on core histones showed that alpha1 are involved in DNA-binding. To understand the molecular basis of NF-Y sequence-specificity, we constructed deletion and swapping mutants, in which the alpha1 of NC2 and archeal HMfB, a bona fide histonic protein, was placed in NF-YB and NF-YC. Our analysis indicates that (i) subunit interactions are normal; (ii) NF-YB-NF-YC and NC2alpha/beta do not form heterodimers and NC2 cannot associate NF-YA. (iii) None of the NF-Y swaps can complex with TBP on a TATA box. (iv) Specific residues, R47 and K49 in NF-YC and N61 in NF-YB, are crucial for CCAAT-binding. We conclude that specificity of the NF-Y trimer is not due to NF-YA only, but stems in part from the contribution of the histone fold alpha1, particularly that of NF-YB.
Zemzoumi
K
K
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Via Celoria 26, Milano, 20133, Italy.
Frontini
M
M
Bellorini
M
M
Mantovani
R
R
D000595:Amino Acid Sequence; D001105:Archaea / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D006657:Histones / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism