Volume 25, Issue 6, Pages 813-823 (March 2007) Insights into Transcription Initiation and Termination from the Electron Microscopy Structure of Yeast RNA Polymerase III  Carlos Fernández-Tornero, Bettina Böttcher, Michel Riva, Christophe Carles, Ulrich Steuerwald, Rob W.H. Ruigrok, André Sentenac, Christoph W. Müller, Guy Schoehn  Molecular Cell  Volume 25, Issue 6, Pages 813-823 (March 2007) DOI: 10.1016/j.molcel.2007.02.016 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Single-Particle Analysis of RNAPIII (A) SDS-PAGE analysis of purified RNAPIII. (B) Partial field of negatively stained RNAPIII used for RCT. (C) Cryo-EM field of ice-vitrified, unstained RNAPIII particles (circle). The contrast is inverted, as employed for subsequent 3D particle analysis. (D) Low-pass filtered raw images of RNAPIII (top row), corresponding averages (middle), and reprojections of the final model in equivalent orientations (bottom). (E) Distribution of projection angles of the 3D reconstruction showing the approximate number of particles in each orientation. For simplicity, only odd classes are plotted. (F) Fourier shell correlation (FSC) function curve. Molecular Cell 2007 25, 813-823DOI: (10.1016/j.molcel.2007.02.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Cryo-EM Structure of RNAPIII (A) Surface representation of the RNAPIII EM density after reconstruction. DNA binding cleft, jaws, clamp, wall, lobe, stalk, saddle, foot, funnel, and NTP entry pore (pore 1) initially identified for RNAPII (Cramer et al., 2001) are indicated on the different views. The presumed position of the active center is marked with a magenta dot. Straight arrows indicate the putative direction of upstream and downstream DNA on the side view. Figure prepared with PyMOL (DeLano, 2002), with a contour level calculated from the molecular mass of the complex and an average protein density of 0.84 Da/Å3. (B) Top views of yeast RNAPII, low-pass filtered to 20 Å by using the coordinates of the 12 subunit crystal structure (Armache et al., 2005; PDB ID code 1WCM), and of the cryonegatively stained EM reconstructions of human RNAPII (Kostek et al., 2006; MSD ID code EMD-1282) and yeast RNAPI (De Carlo et al., 2003). Molecular Cell 2007 25, 813-823DOI: (10.1016/j.molcel.2007.02.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Structural Differences between RNAPII and III (A) Final manual fitting of RNAPII crystal structure (Armache et al., 2005; PDB ID code 1WCM) into the reconstructed RNAPIII density. Domains of RNAPII that fall outside the EM density are labeled MD1–MD6 (Table 2). For clarity, the main structural elements are indicated outside the density; for precise positioning, refer to Figures 2A and 3B. Mg2+ and Zn2+ ions appear as magenta- and yellow-colored dots, respectively. (B) Difference map between the RNAPIII cryo-EM structure and the RNAPII crystal structure. Additional density features (green) in the RNAPIII reconstruction are labeled AD1–AD7 (Table 2). The N-terminal domain of Rpb9, positioned according to the fitting in (A), is in yellow. The putative position of the active center is marked with a magenta dot. (C) Schematic representation and color code of RNAPII subunits, with corresponding RNAPIII subunits in parenthesis (Table 1). Molecular Cell 2007 25, 813-823DOI: (10.1016/j.molcel.2007.02.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Close-Up View of the RNAPIII Stalk (A) In the RNAPII model fitted into the EM reconstruction, stalk subunits Rpb4/Rpb7 were replaced by the RNAPIII C17/C25 subcomplex (Jasiak et al., 2006) by superposing subunit C25 onto Rpb7. Squares indicate the position of two insertions in C17/C25 with respect to Rpb4/Rpb7 (Figure S2) that are disordered in the RNAPIII-stalk crystal structure. (B) Stalk subunits Rpb4/Rpb7 in the RNAPII model as depicted in Figure 3A. Squares indicate the position of two insertions in the RNAPIII subunits C17/C25 (see above). Molecular Cell 2007 25, 813-823DOI: (10.1016/j.molcel.2007.02.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Immunoaffinity Labeling of RNAPIII Subunits C82 and C34 (A) Localization of the anti-C34 antibodies in binary or ternary complexes by comparison of filtered, aligned particle images with bound antibody (upper row) against 3D surfaces of RNAPIII modeled together with bound antibody in the corresponding orientations (lower row). Boxed numbers in the upper row correspond approximately to the views shown in (C). The positions of additional densities AD1-3 are indicated. (B) Localization of the anti-C82 antibodies in binary complexes. Comparison of filtered, aligned particle images with bound antibody (upper row) against 3D surfaces as detailed in (A). (C) Orientations 1–4 of RNAPIII as present in (A) and (B). The positions of the additional densities AD1–3 and the main structural elements are indicated. (D) Summary of the immunolabeling results showing the putative position of subunits C82 and C34 in the RNAPIII complex. The N-terminal domain of TFIIB (Bushnell et al., 2004) superimposed onto the RNAPII structure in Figure 3A is depicted as orange surface. Thr69 in RNAPII subunit Rpb1 is indicated as a black dot. Molecular Cell 2007 25, 813-823DOI: (10.1016/j.molcel.2007.02.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 RNAPIII Transcription Model Model of transcribing RNAPIII showing the putative positions of RNAPIII-specific subunits, in two different orientations. DNA coding and noncoding strands and RNA as observed in transcribing RNAPII (Kettenberger et al., 2004; PDB ID code 1Y1W) are depicted in blue, cyan, and red, respectively. The presumed path for newly synthesized RNA is indicated with a red dotted line. The relationships between the RNAPIII subunits and TFIIIB subunit Brf1 during transcription are schematized with arrows. Molecular Cell 2007 25, 813-823DOI: (10.1016/j.molcel.2007.02.016) Copyright © 2007 Elsevier Inc. Terms and Conditions