Binding of TFIIB to RNA Polymerase II Hung-Ta Chen, Steven Hahn  Molecular Cell  Volume 12, Issue 2, Pages 437-447 (August 2003) DOI: 10.1016/S1097-2765(03)00306-X

Figure 1 TFIIB Mutants Used in the Photocrosslinking (A) TFIIB domains. The S. cerevisiae zinc ribbon and the core domain are colored light blue and green, respectively. Red arrows below the zinc ribbon indicate the five positions for cys mutation. A highly conserved sequence block (pink, CB) is located adjacent to the ribbon. The two cyclin-like repeats in the core domain are denoted by arrows. Four cysteine residues in the core domain are mutated to either alanine or serine as indicated. The TFIIB mutants used in photocrosslinking (but not the FeBABE studies) also contained two copies of the Flag epitope at the C terminus (orange, 2X FLAG). The hydroxylamine cleavage sequence “NG” and the predicted size of each peptide cleavage fragment is listed in Daltons. (B) Functional surface of the ribbon and position of cys substitutions. The TFIIB ribbon domain is shown with sidechains, that show no phenotype when mutated colored yellow or green (Hahn and Roberts, 2000), sidechains that affect function when mutated colored red, and residues mutated individually to cys colored green. (C) Hydroxylamine cleavage of PEAS labeled TFIIB mutants. SDS-PAGE of the hydroxylamine cleavage is revealed by both Coomassie blue stain and autoradiography. Specific radioactivity of the N-terminal peptide from hydroxylamine cleavage is listed in parentheses below each peptide. The specific 125I radioactivity of the N-terminal peptide is derived from the ratio of the label incorporated and volume of the Coomassie staining, both determined by IQMac software (Molecular Dynamics), and is normalized to the wild-type, non-cys substituted zinc ribbon. Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)

Figure 2 Transcription Activity of PEAS Substituted TFIIB Variants and Photocrosslinking. (A) The transcription activity assayed by primer extension of single round transcription using a DNA template containing the HIS4 promoter and an upstream Gal4 binding site. The nuclear extract used in the assay is from a yeast strain containing TFIIB with a zinc ribbon temperature sensitive mutation and is inactive in the transcription assay without addition of a functional TFIIB (-). Both labeled and unlabeled cys-substituted TFIIB mutants are assayed as indicated. In comparison, the transcription activities using completely wild-type TFIIB (sua7) and non-cys TFIIB (WT) are also shown. Relative transcription activity is listed above each lane. (B and C) Autoradiography of photocrosslinking. PICs were formed using the indicated TFIIB variant linked to PEAS and isolated using an immobilized promoter template. Samples were treated with UV and DTT as indicated and products separated by SDS PAGE. Asterisks mark specific crosslinked polypeptides. M is the 14C molecular weight marker. (D) Rpb1 and Rpb2 are identified as the 180 and 130 kDa crosslinked polypeptides using epitope-tagged Rpb1 and Rpb2. Nuclear extracts from strains containing Flag-tagged Rpb1 and Rpb2 were used in the crosslinking assay as indicated. The same mobility shifts are also observed in the experiments with R37C and E40C (not shown). The unidentified 150 kDa crosslinked polypeptide is indicated by ? Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)

Figure 3 Transcription Activity of FeBABE Substituted TFIIB Variants and Directed Hydroxyl Radical Probing (A) Same assay as described in Figure 2A, single round transcription activities for FeBABE substituted TFIIB variants. (B and C) Cleavage fragments of Rpb1 with Flag attached in the C terminus (Rpb1_Flag, in [B]) or inserted after residue 45 (Rpb1_45Flag, in [C]) were visualized by Western blotting with Flag antibody such that only fragments containing the Flag tag are observed. Cys-substituted TFIIB mutants as indicated were conjugated with FeBABE and used in the PIC formation/directed hydroxyl radical probing (lanes 3–7 and 10–14). Negative control experiment (-) contains no added FeBABE-TFIIB and thus only endogenous TFIIB in the nuclear extract is present in the assay (lanes 1 and 8). Brackets indicate the specific cleavage fragments. The arrows point to full-length Rpb1. M, molecular size marker. (D) Specific cleavage fragments of C-terminal Flag-tagged Rpb2 (Rpb2_Flag). Arrow indicates full-length Rpb2. Cleavage products in the lower molecular weight range are shown with higher sensitivity due to their low intensity (lower panel). Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)

Figure 4 In Vitro Translated Peptide Fragments Used as Molecular Standards for Calculating FeBABE Cleavage Sites (A and B) In vitro translated peptide fragments of the N-terminal region of Rpb1_45Flag and C-terminal region of Rpb2_Flag analyzed by SDS PAGE. The black blocks indicate positions of the Flag sequence in Rpb1_45Flag and Rpb2_Flag. Each sample is represented by the bar along with the corresponding molecular size (in parentheses) and the last (Rpb1_45Flag) or first (Rpb2_Flag) residue. For each lane in (A), only the slower polypeptide is from the endogenous AUG translation start site. (C) Calibration curves for determining the amino acid numbers of FeBABE cleavage fragments. Each calibration curve is derived from a 4th order polynomial function using the sizes (log(MW)) of in vitro translated peptides and their corresponding amino acid numbers in (A) and (B). Locations of the FeBABE cleavage fragments on the calibration curves are shown with triangles. Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)

Figure 5 FeBABE Cleavage Sites and Model for Ribbon Domain Interaction (A) The calculated FeBABE cleavage sites are mapped to the surface of the Pol II form 1 structure (Cramer et al., 2001). A nine-residue segment centered on the calculated cleavage site is colored as light blue (weak cleavage) or dark blue (strong/medium cleavage). The structural region surrounded by the clamp, wall, and dock domains is indicated and also contains two grooves labeled 1 (the RNA exit channel) and 2. (B) Model for interaction of the ribbon domain with Pol II. The ribbon backbone is shown in yellow and, in the model, interacts with a surface of the dock domain. Position of residue R412 of Rpb1 is colored in red. Two different views with a rotation of 25° are provided. The N and C termini of the ribbon are indicated. The magenta sphere represents the zinc ion. The surface of Rpb6 subunit is colored in orange. (A) and (B) were generated using GRASP (Nicholls et al., 1991). Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)

Figure 6 ClustalX Sequence Alignment of the Dock Domains in Eukaryotic, Archaeal, and Bacterial RNA Polymerases Top, schematic of the dock domain and secondary structure elements in the Pol II dock. The dock domain sequence is located between the highly conserved homology blocks C and D, which form the active site in the structure. Bottom, highlighted are the residues identical or conserved in two out of five classes of polymerases. The green bracket indicates the dock residues predicted by modeling to be in contact with the TFIIB ribbon domain. The asterisk indicates residue R412 in yeast Rpb1. The blue bar indicates residues 416-420 of yeast Rpb1. Dock domain sequences listed are from organisms: yeast, Saccharomyces cerevisiae; pombe, Schizosaccharomyces pombe; fly, Drosophila melanogaster; worm, Caenorhabditis elegans; hum, Homo sapiens; Pyrfu, Pyrococcus furiosus; Arcfu, Archaeoglobus fugidus; Metth, Methanobacterium thermoautotrophicum; Halsa, Halobacterium salinarium; Ecoli, Escherichia coli; and Taq, Thermus aquaticus. Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)

Figure 7 Dock Domain Mutations Affect the Pol II-TFIIB Interaction (A) Western blot of coimmune precipitated proteins using Anti-Flag affinity gel. Nuclear extracts containing C-terminal Flag tagged Rpb1 (Flag) with no mutation (WT), R412H, or R412A in the dock domain were used as indicated. A control experiment (WT, no Flag) uses nuclear extract containing wild-type Rpb1 without Flag tag. (B) Western blot of Pol II immobilized template assay. As in (A), same set of four nuclear extracts are used in the immobilized template-PIC formation experiment. The relative intensities of Rpb1 are listed below. (C) In vitro transcription activities. Immobilized template single-round transcription with the four nuclear extracts was performed as described at www.fhcrc.org/labs/hahn. The relative transcription activities are listed below. Molecular Cell 2003 12, 437-447DOI: (10.1016/S1097-2765(03)00306-X)