Synergistic Transcriptional Activation by TATA-Binding Protein and hTAF(II)28 Requires Specific Amino Acids of the hTAF(II)28 Histone Fold

Coexpression of the human TATA-binding protein (TBP)-associated factor 28 (hTAF(II)28) with the altered-specificity mutant TBP spm3 synergistically enhances transcriptional activation by the activation function 2 of the nuclear receptors (NRs) for estrogen and vitamin D(3) from a reporter plasmid containing a TGTA element in mammalian cells. This synergy is abolished by mutation of specific amino acids in the α2-helix of the histone fold in the conserved C-terminal region of hTAF(II)28. Critical amino acids are found on both the exposed hydrophilic face of this helix and the hydrophobic interface with TAF(II)18. This α-helix of hTAF(II)28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1′ α-helix of TBP either reduces or increases interactions with hTAF(II)28. The mutations which reduce interactions with hTAF(II)28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAF(II)28 is defective in its ability to synergistically enhance activation by NRs. However, this TBP mutant supports activation by other activators and is thus specifically defective for its ability to synergize with hTAF(II)28.