Distinct domains of hTAF(II)100 are required for functional interaction with transcription factor TFIIFβ (RAP30) and incorporation into the TFIID complex

Abstract

TFIID is the DNA binding component of the RNA polymerase II transcriptional machinery and is composed of the TATA binding protein (TBP) and TBP-associated factors (TAF(II)s). Here we report the characterization of a new human TAF, hTAF(II)100, which is the human homologue of Drosophila TAF(II)80 and yeast TAF(II)90. hTAF(II)100 interacts strongly with hTAF(II)250, hTAF(II)55 and hTAF(II)28, less with hTAF(II)20 and hTAF(II)18, weakly with TBP and not at all with ΔNTAF(II)135 and hTAF(II)30. Deletion analysis revealed that the C-terminal half of hTAF(II)100, which contains six WD-40 repeats, is not required for incorporation into the TFIID complex. Our results suggest that hTAF(II)100 can be divided into two domains, the N-terminal region responsible for interactions within the TFIID complex and the C-terminal WD repeat-containing half responsible for interactions between hTAF(II)100 and other factors. An anti-hTAF(II)100 antibody, raised against a C-terminal epitope, selectively inhibited basal TFIID-dependent in vitro transcription and the specific interaction between hTAF(II)100 and the 30 kDa subunit of TFIIF (RAP30). We demonstrate that the hTAF(II)100-TFIIF interaction supports pre-initiation complex formation in the presence of TFIID. Thus, this is the first demonstration that a TAF(II) functionally interacts with a basal transcription factor in vitro.