Abstract
The general transcription factor TFIID is a multiprotein complex consisting of the TATA box-binding protein and multiple TATA box-binding protein-associated factors (TAF(II)s). The central domain of human TAF(II)130 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 hTAF(II)130s with alterations in TAF(II)-activator interaction. Here we characterize functionally an hTAF(II)130 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 hTAF(II)130 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 (Delta9) surrounding Phe(311) abolished the interaction of hTAF(II)130 with Sp1. Overexpression of hTAF(II)130Q1/Q2 and Q1-Q4 strongly inhibited Sp1-dependent transcriptional enhancement in transient transfection assays, whereas expression of either F311S or Delta9 only partially suppressed Sp1-mediated activation. Thus, a short hydrophobic sequence motif encompassing Phe(311) in hTAF(II)130 represents a critical surface with which Sp1B interacts to activate transcription.
Highlights
The initiation of transcription in eukaryotes is regulated by a network of protein-protein interactions between sequencespecific transcription factors, their cofactors, and components of the general transcription machinery
To examine which amino acid residues within the Q1/Q2 domain are important for mediating Sp1B and cAMP-response element binding protein (CREB)-N interactions, we constructed a number of Q1/Q2 point mutants and tested them for their ability to interact with Sp1B and CREB-N in yeast two-hybrid assays (Fig. 1)
The reduced activity was not due to decreased expression of the mutant protein because its level of expression was comparable with that of the wild type. These results suggest that Phe311 plays an important role in allowing hTAFII130 to interact with the Sp1B and CREB-N activation domains
Summary
The initiation of transcription in eukaryotes is regulated by a network of protein-protein interactions between sequencespecific transcription factors, their cofactors, and components of the general transcription machinery (reviewed in Refs. 1–5). HTAFII130 mutants exhibiting increased or decreased interaction with Sp1B or CREB-N (higher or lower -galactosidase activity compared with wild type) were sequenced to determine the mutated residues.
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