A Shared Surface of TBP Directs RNA Polymerase II and III Transcription via Association with Different TFIIB Family Members  Xuemei Zhao, Laura Schramm, Nouria Hernandez, Winship Herr  Molecular Cell  Volume 11, Issue 1, Pages 151-161 (January 2003) DOI: 10.1016/S1097-2765(02)00797-9

Figure 1 Model Promoters and TFIIB Family Members (A) Illustration of the basal promoter elements in the AdML, U1, U6, and VAI promoters. PSE, proximal sequence element; POL., RNA polymerase. (B) Schematic structure of the human TFIIB family members TFIIB, Brf1, and Brf2 (Schramm and Hernandez, 2002). The location of the structured zinc (Zn) ribbon (green) and core domain (blue) of TFIIB and that of the corresponding regions in the other proteins are indicated. The purple boxes in Brf1 indicate conserved regions II and III between human and yeast Brf1. Molecular Cell 2003 11, 151-161DOI: (10.1016/S1097-2765(02)00797-9)

Figure 2 Human TBP-Dependent Transcription Parallel in vitro transcription reactions were performed from the (A) AdML, (B) U1, and (C) U6 promoters in the absence or presence of human TBP as indicated above each lane with an equal amount of untreated (lanes 1 and 2) or mock- (lanes 3 and 4) or TBP- (lanes 5 and 6) depleted HeLa cell extract. (D) In vitro transcription reactions performed with the VAI promoter in the absence or presence of human TBP or Brf1 as indicated above each lane, with an equal amount of untreated (lanes 1–4), or mock- (lanes 5–8) or TBP- (lanes 9–12) depleted HeLa cell extract. Except for HeLa cell nuclear extract used for the U1 transcription reactions, the reactions were performed with whole-cell extract. CT, correct transcript; IC, internal control for sample preparation and RNase T1 digestion; *, nonspecific RNase protected fragment from the probe preparation. Molecular Cell 2003 11, 151-161DOI: (10.1016/S1097-2765(02)00797-9)

Figure 3 Mutations on the Surface of the Human TBPCORE in Full-Length TBP (A) Schematic representation of human TBP. The arrows indicate two imperfect repeats. (B) Downstream or front view of the human TBPCORE (white) bound to the TATA box-containing DNA (green) (Nikolov et al., 1996). (C and D) Front and back views, respectively, of the molecular surface representations of the human TBPCORE (white) bound to DNA (green) (Nikolov et al., 1996) together with superimposed core regions of yeast TFIIA (orange) (Geiger et al., 1996; Tan et al., 1996) and human TFIIB (cyan) (Nikolov et al., 1995). (E–H) Front, back, top, and bottom views, respectively, of the human TBPCORE-TATA box complex with amino acid substitutions colored as (i) mauve on the top and sides of the TBPCORE, (ii) orange on the TFIIA-interaction surface, (iii) cyan on the TFIIB interaction surface, and (iv) green on the bent TATA box recognition surface. Molecular Cell 2003 11, 151-161DOI: (10.1016/S1097-2765(02)00797-9)

Figure 4 Effects of Mutations on the Human TBPCORE in Full-Length TBP in Basal Transcription from Four Model Promoters (A) Effects of four categories of substitutions—Top and Side (lanes 5–10), TFIIA (lanes 11–15) and TFIIB (lanes 16 and 17) interaction surfaces, and the DNA binding surface (lanes 18–22)—on the human TBPCORE domain of full-length TBP in transcription from the (i) AdML, (ii) U1, (iii) U6, and (iv) VAI promoters. Parallel in vitro transcription reactions were performed in the presence of purified human wild-type (lanes 2, 4, and 23) or mutant (lanes 5–22) TBP molecules in mock- (lanes 1 and 2) or TBP- (lanes 3–23) depleted HeLa cell extract. VAI transcription was performed in the presence of supplemented purified Brf1. The arrowhead indicates an additional U1 transcript. (B) Front, top, back, and bottom molecular surface representations of the TBPCORE with residues colored according to the category of substitution as in Figure 3 (top row) or their respective activities compared to wild-type TBP (100%) in transcription from the four model promoters (bottom four rows): blue, >50%; yellow, 15%–50%; and red, <15%. Residues for which multiple substitutions were assayed: the most severe effect is shown. Molecular Cell 2003 11, 151-161DOI: (10.1016/S1097-2765(02)00797-9)

Figure 5 The Second Stirrup Region of the Human TBPCORE in Full-Length TBP Interacts Selectively with the Human TFIIB Family Proteins Electrophoretic mobility retardation analysis of the interactions between the human wild-type and second stirrup region mutant TBP molecules with or without TFIIB (lanes 1–12), Brf2 (lanes 13–24), or Brf1 (lanes 25–36) as indicated above each lane on the TATA box-containing (A) AdML and (B) U6 promoter DNAs. The open and filled circles to the left of the TBP-TATA box complexes indicate the unbent TBPFL and bent TBPFL* complexes, respectively. The blue and red triangles to the left of the TFIIB family-TBP-TATA box complexes indicate the wild-type and mutant altered mobility complexes, respectively. *, irrelevant band. Molecular Cell 2003 11, 151-161DOI: (10.1016/S1097-2765(02)00797-9)

Figure 6 The Second Stirrup Region of the Human TBPCORE in Full-Length TBP Does Not Interact with SNAPC Electrophoretic mobility retardation analysis of the interactions between the human wild-type (lanes 3 and 4) and second stirrup region mutant (lanes 5–12) TBP molecules with (even numbered lanes) and without (odd numbered lanes) purified SNAPC on the TATA box and PSE of the human U6 promoter. The unbent (TBPFL) and bent (TBPFL*) TBP-TATA box complexes, and SNAPC-DNA and SNAPC-TBP-DNA complexes are indicated to the left of the figure. Molecular Cell 2003 11, 151-161DOI: (10.1016/S1097-2765(02)00797-9)