CCAAT BINDING NF-Y-YBP 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 the ir binding proteins, TBP and NF-Y, are extremely conserved in evolutio n. NF-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, all nec essary for DNA binding. NF-YB and NF-YC contain a putative histone-lik e motif, a domain also present in TBP-associated factors (TAF(II)s) an d in the subunits of the transcriptional repressor NC2. Immunopurifica tion of holo-TFIID with anti-TBP and anti-TAF-(II)100 antibodies indic ates 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 sh owing that: (i) NF-YB and NF-YC interact with TBP in solution, both se parately and once bound to each other; (ii) short stretches of both NF -YB and NF-YC located within the evolutionary conserved domains, adjac ent to the putative histone fold motifs, are necessary for TBP binding ; (iii) TBP single amino acid mutants in the HS2 helix, previously sho wn to be defective in NC2 binding, are also unable to bind NF-YB and N F-YC.