The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF II 170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF II 170. We have defined the TBP interaction domain of TAF II 170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP AS ) containing a triple mutation in the concave surface is defective for binding the TAF II 170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF II 170 residues 290 to 381 can inhibit the interaction between Drosophila TAF II 230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF II 170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP AS mutant is less sensitive to TAF II 170 inhibition. Collectively, our results support a mechanism in which TAF II 170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.Initiation of mRNA synthesis by RNA polymerase (pol) II requires the basal transcription factors TFIID, TFIIB, TFIIE, TFIIF, and TFIIH. Recognition of the TATA box within core pol II promoters by TFIID initiates the assembly of a functional pol II preinitiation complex. TFIID is a multisubunit complex consisting of TATA-binding protein (TBP) and at least 10 TBP-associated factors (TAF II s) ranging in size from 15 to 250 kDa. TAF II s play several roles in transcriptional regulation, serving as coactivators, promoter selectivity factors, or enzymes that modify surrounding transcriptional proteins (3). The TBP subunit recognizes the TATA box forming the TBP-TATA complex (24). Structural studies of the TBP-TATA complex indicate that the ␤ sheets of TBP form a concave undersurface which contacts the minor groove of the TATA box (28,29).Of relevance to this study is that the largest TAF II subunit of TFIID (dTAF II 230 in Drosophila, hTAF II 250 in humans, and yTAF II 145/130 in yeast), can inhibit TBP binding to the TATA box (11,31,33). dTAF II 230 achieves this by interacting with the concave DNA binding surface of TBP (see references cited above). Residues 2 to 81 (called TAND I) undergo a disorderto-order transition upon interaction with TBP. The induced structure of TAND I mimics the structure of the TATA box when bound to TBP (34). A second N-terminal inhibitory domain (TAND II; residues 82 to 156) blocks the TBP-TFIIA interaction by competitively binding to helix 2 (H2) on the convex surface of TBP (10). The orthologous TAF II subunits from yeast and humans contain regions which are functionally equivalent to TAND I and TAND II despite a low degree of primary sequence similarity (33).TAF II -contain...