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 en hances transcriptional activation by the activation function 2 of the nucle ar receptors (NRs) for estrogen and vitamin D-3 from a reporter plasmid con taining a TGTA element in mammalian cells. This synergy is abolished by mut ation of specific amino acids in the alpha 2-helix of the histone fold in t he conserved C-terminal region of hTAF(II)28. Critical amino acids are foun d on both the exposed hydrophilic face of this helix and the hydrophobic in terface with TAF(II)18. This alpha-helix of hTAF(II)28 therefore mediates m ultiple interactions required for coactivator activity. We further show tha t mutation of specific residues in the H1' alpha-helix of TBP either reduce s or increases interactions with hTAF(II)28. The mutations which reduce int eractions with hTAF(II)28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAF(II)28 is defective in its a bility to synergistically enhance activation by NRs. However, this TBP muta nt supports activation by other activators and is thus specifically defecti ve for its ability to synergize with hTAF(II)28.