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.