Genetic Dissection of hTAFII130 Defines a Hydrophobic Surface Required for Interaction with Glutamine-rich Activators

The general transcription factor TFIID is a multiprotein complex consisting of the TATA box-binding protein and multiple TATA box-binding protein-associated factors (TAFIIs). The central domain of human TAFII130 contains four glutamine-rich regions Q1-Q4 that interact with transcriptional activators such as Sp1 and CREB and mediate activation. We screened in yeast random point mutations introduced into Q1-Q4 against the Sp1 activation domain and obtained a distinct set of hTAFII130s with alterations in TAFII-activator interaction. Here we characterize functionally an hTAFII130 mutant containing a phenylalanine to serine change at position 311 (F311S) that is compromised in its ability to associate with Sp1B and CREB-N activation domains. Substitution of phenylalanine with tyrosine but not with isoleucine or tryptophan also reduced hTAFII130 interaction, suggesting that the hydrophobic character rather than the specific amino acid at this position is a key determinant of interaction. Deletion of nine amino acids (Δ9) surrounding Phe311 abolished the interaction of hTAFII130 with Sp1. Overexpression of hTAFII130Q1/Q2 and Q1-Q4 strongly inhibited Sp1-dependent transcriptional enhancement in transient transfection assays, whereas expression of either F311S or Δ9 only partially suppressed Sp1-mediated activation. Thus, a short hydrophobic sequence motif encompassing Phe311 in hTAFII130 represents a critical surface with which Sp1B interacts to activate transcription.