NF-Y histone fold alpha1 helices help impart CCAAT specificity

Area of Science:

• Molecular Biology
• Gene Regulation
• Protein Structure

Background:

• Nuclear Factor Y (NF-Y) is a trimeric transcriptional activator essential for gene expression.
• NF-Y's subunits NF-YB and NF-YC share homology with histone fold motifs, similar to NC2alpha/beta, a repressor complex.
• Histone fold alpha1 helices are implicated in DNA binding, suggesting a role in sequence specificity.

Purpose of the Study:

• To elucidate the molecular basis of NF-Y's sequence-specific DNA binding.
• To investigate the contribution of histone fold motifs to NF-Y's specificity for CCAAT boxes.

Main Methods:

• Construction and analysis of deletion and swapping mutants of NF-YB, NF-YC, NC2, and archeal HMfB.
• Assessment of subunit interactions and complex formation with TBP (TATA-binding protein).
• Identification of critical residues involved in CCAAT-binding.

Main Results:

• Subunit interactions within NF-Y and NC2 complexes were maintained in mutants.
• NC2 did not form heterodimers with NF-YB/NF-YC and could not associate with NF-YA.
• Mutated NF-Y complexes failed to bind TBP on a TATA box.
• Specific residues (R47, K49 in NF-YC and N61 in NF-YB) were identified as crucial for CCAAT binding.

Conclusions:

• NF-Y's sequence specificity is not solely determined by the NF-YA subunit.
• The histone fold alpha1, particularly from NF-YB, contributes significantly to NF-Y's CCAAT-binding specificity.
• This finding highlights the role of histone-like structures in the DNA-binding specificity of transcription factors.