TAF_II170 Interacts with the Concave Surface of TATA-Binding Protein To Inhibit Its DNA Binding Activity

Abstract

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...

NC2α Interacts with BTAF1 and Stimulates Its ATP-Dependent Association with TATA-Binding Protein

“…On the other hand, BTAF1 and Mot1p proteins contain dATPase activity, which is involved in the dissociation of TBP from DNA in an ATPdependent stroke. This activity can explain their negative effect on transcription (5,34,36). In accordance with a dual role of Mot1p in transcription, mRNA expression profiling and mutational analyses indicate that Mot1p affects transcription both positively and negatively (1,6,10,12,15,26,27,39).…”

“…Plasmids encoding LexA fusions of BTAF1 fragments used in a yeast two-hybrid screen were described previously (36). pJG-NC2␣ and pJG-NC2␤ were obtained by inserting NC2␣ and NC2␤ coding sequences flanked by EcoRI sites into EcoRI-digested pJG4-5.…”

“…pGST-BTAF1(505-891) was created by inserting the appropriate BTAF1 cDNA fragment into pGEX-2T with the modified polylinker. pGST-⌬TFIIS was described previously (36). Plasmid pTM3 containing cDNA coding for BTAF1 mutant K1297A was constructed using PCR-based mutagenesis and used to construct a BTAF1 K1297A-expressing vaccinia virus as described previously (34).…”

“…Escherichia coli BL21(DE3) strains expressing an appropriate protein or pair of proteins were cultured on a 2-liter scale and lysed by lysozyme treatment as described previously (36). To obtain NC2(his␣/␤), NC2(␣/his␤), NC2(GST␣/his␤), and NC2(his␣/GST␤) complexes, bacteria were transformed with the appropriate pair of pET-and pACYC-based plasmids and grown in the presence of 50 l of ampicillin/ml and 30 g of chloramphenicol/ml.…”

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

“…Analyses of different DNA templates, Mot1 mutants, footprinting, and cross-linking have established that although Mot1 has no detectable DNA binding on its own, in the absence of ATP, it forms a ternary complex with TBP and DNA via interaction with residues in TBP and contact with base pairs within an ϳ17-bp region upstream of the TATA box (27)(28)(29). The length of the upstream DNA region is about what would be expected if the Mot1 ATPase docks onto DNA in a manner similar to that of a highly related ATPase, SsoRad54 (30).…”