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Crystal structure of the 14-subunit RNA polymerase I

Nature volume 502, pages 644–649 (2013)Cite this article

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Abstract

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we present the crystal structure of Pol I from Saccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a transcription elongation factor TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to α-amanitin. The A49–A34.5 heterodimer embraces subunit A135 through extended arms, thereby contacting and potentially regulating subunit A12.2.

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Figure 1: Crystal structure of yeast RNA polymerase I.
Figure 2: An extended loop mimics the transcription bubble inside the DNA-binding cleft.
Figure 3: Stalk anchoring in Pol I, Pol II and archaeal Pol.
Figure 4: Pol I subunit A12.2 extends from the jaw to the active site.
Figure 5: Anchoring of the A49–A34.5 heterodimer onto the Pol I core.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors of the three related Pol I crystal forms have been deposited at the Protein Data Bank under accession numbers 4C3H, 4C3I and 4C3J.

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Acknowledgements

We thank H. Grötsch for preparing the loop deletion yeast strain, and G. von Scheven and A. Scholz for technical assistance. We are also grateful to C. Vonrhein, G. Bricogne, S. Glatt and A. Romero for advice and discussions. We thank staff from the European synchrotrons SOLEIL, DESY, ESRF and SLS, where data were collected during different stages of the project. In particular, we thank A. Thompson for access and support at beamline Proxima 1 (Soleil) and T. Schneider and G. Bourenkov at beamline P14 (PETRA III). We also acknowledge support by the EMBL Heidelberg Protein Expression and Purification, Proteomics Core Facilities and Crystallization Platform, and the ‘Fermentation et culture de microorganisms’ (IFR88, CNRS). We are grateful to M. Bauzan, E. Poilpre and J. Scheurich for yeast fermentation. M.M.-M. and U.J.R. were supported by EMBO Long-Term fellowships, M.M.-M. by the Marie-Curie fellowship (FP7-PEOPLE-2011-IEF 301002), N.M.I.T. by a Fundación Futuro fellowship, F.M.R. by an ESF/CSIC funded JAE-DOC contract and T.G. by the Volkswagen Stiftung via the Niedersachsenprofessur of Prof. G. M. Sheldrick. This work was also partly funded by grant BFU2010-16336 of the Spanish Ministry of Science.

Author information

Author notes

  1. Ulrich Steuerwald

    Present address: Present address: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.,

  2. Carlos Fernández-Tornero and María Moreno-Morcillo: These authors contributed equally to this work.

Authors and Affiliations

  1. Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain ,

    Carlos Fernández-Tornero, Nicholas M. I. Taylor & Federico M. Ruiz

  2. European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany ,

    María Moreno-Morcillo, Umar J. Rashid, Ulrich Steuerwald & Christoph W. Müller

  3. Department of Structural Chemistry, Georg-August-University, Tammannstraße 4, 37077 Göttingen, Germany,

    Tim Gruene

  4. SOLEIL Synchrotron, L’Orme des Merisiers Saint Aubin, 91192 Gif-sur-Yvette, France ,

    Pierre Legrand

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Contributions

C.F.-T. and C.W.M. initiated the project. C.F.-T. and U.S. established the Pol I purification and obtained Pol I crystals. C.F.-T., M.M.-M. and U.J.R. further improved the Pol I crystals, and collected data and obtained experimental phase information with the help of N.M.I.T., T.G. and P.L. C.F.-T., M.M.-M., N.M.I.T., F.M.R., T.G. and P.L. carried out the crystallographic analysis and model refinement. C.F.-T., M.M.-M. and C.W.M. wrote the manuscript with input from the other authors.

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Correspondence to Carlos Fernández-Tornero or Christoph W. Müller.

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Extended data figures and tables

Extended Data Figure 1 Electron densities of different regions of the Pol I structure.

a, Helix in the cleft of subunit A190. Subunits are coloured according to the code given in Fig. 1a. In a–d, σA-weighted electron densities contoured at 1σ are depicted in blue. b, Interaction between subunit Rpb8 (green) and subunit A190 (grey). c, Linker between the two Zn ribbon domains in subunit A12.2. d, A49–A34.5 heterodimer and the anchoring onto the Pol I core by the A34.5 hook and the A49 linker region. e, Anomalous difference Fourier map (purple) calculated from partially selenomethionine-substituted Pol I contoured at 3σ (Extended Data Table 1). In the A49–A34.5 heterodimer two selenium peaks correspond to A34.5 Met 80 and Met 107. f, Anomalous difference Fourier map (purple) showing selenomethionine positions contoured at 3σ. In total, 90 out of 100 expected selenium positions were located within a distance of less than 2.3 Å to corresponding methionine residues.

Extended Data Figure 2 Comparison between Pol I, Pol II and archaeal Pol.

a, Crystal structures of yeast Pol I and Pol II and the archaeal Pol are represented in the same orientation using the same colour code. Whereas the overall organization is conserved, additional subunits such as the A49–A34.5 heterodimer in Pol I and Rpo13 in Archaea are also present. The archaeal Pol lacks the orthologue of subunit A12.2 in Pol I (or Rpb9 in Pol II). The relative position of the stalk also varies between the three RNA polymerases. b, Crystal structures of the individual subunits varying between the three enzymes are depicted. The same colour code for corresponding or identical subunits in the three RNA polymerases is used.

Extended Data Figure 3 Opening of the cleft varies among different Pol I structures and between Pol I and Pol II.

a, Middle panel: front view of the Pol I structure in crystal form C2-100 (see Extended Data Table 1). The complex is divided into two modules. Module 1 (red) is formed by the major part of subunit A190 (without the pore 1, funnel and jaw domains), the C terminus of A135, Rpb5, Rpb6, Rpb8 and the stalk subunits, whereas module 2 (blue) comprises the remaining A135 domains, the pore 1, funnel and jaw domains of A190, AC40–AC19, Rpb10, Rpb12, A12.2 and the A49–A34.5 heterodimer. These modules are held by three hinges in A190 (active site–pore 1 connection, bridge helix and jaw–cleft connection) and one hinge in A135 (hybrid binding–anchor connection) as indicated in Fig. 1b. Pol I structures obtained in crystal forms C2-90 (left panel) and C2-93 (right panel) were superimposed with the one obtained in crystal form C2-100 taking module 2 as reference. Differences between the different crystal forms in the cleft aperture and the tilting of the mobile modules are indicated. b, Pol II structure (Protein Data Bank accession 1WCM) is superimposed onto Pol I (C2-100) using module 1 as reference. In comparison, the Pol II cleft is closed by 10 Å and the modules rotate 15.6°. c, Schematic representation of Pol I and Pol II showing the mobility between Pol I modules, as well as the conformation of the stalk and the clamp. Colour coding is as in b, with the exception of the Pol II stalk, which is coloured deep orange. d, Conformation of the bridge helix of the bacterial Thermus thermophilus polymerase (bacPol, pink, Protein Data Bank accession 1IW7), Pol I (green) and Pol II (yellow, Protein Data Bank accession 1WCM). In addition, the trigger loop is shown for the Thermus thermophilus polymerase, where it is ordered, and as dotted lines for Pol I and Pol II, where it is disordered. e, Sequence alignment of the bridge helix of Saccharomyces cerevisiae and Homo sapiens Pol I, Pol II and Pol III, archaeal Methanococcus jannaschii and Sulfolobus solfataricus, and the bacterial Escherichia coli and Thermus thermophilus polymerases. The secondary structure of the Pol I bridge helix is shown above the alignment. In Methanococcus jannaschii, site-directed mutations Q823D and S824P in subunit A′ lead to increased transcriptional activity.

Extended Data Figure 4 Elongation complex.

a, Cartoon representation of a model of Pol I in complex with an elongation bubble, generated by superposition of the Pol II elongation complex crystal structure (Protein Data Bank accession 1Y1W) using the largest subunit as reference. Whereas Pol II is not shown, the coding and non-coding DNA strands are depicted in blue and cyan, respectively, and the RNA in red. The main Pol I elements putatively involved in nucleic acid interaction appear in different colours, whereas the rest of the Pol I structure is shown in light grey. b, Proposed rearrangements in elongating Pol I (coloured elements) in analogy with Pol II (grey elements). Closure of the cleft is expected to approach the wall (tan), the hybrid binding domain (dark red) and the fork loop 2 (orange) to the bubble, as well as to fold the bridge helix (green). Closure of the fork loop 1 (pink) and opening of the lid loop (cyan) would also be required.

Extended Data Figure 5 The DNA-mimicking loop of Pol I forms a mobile element.

a, σA-weighted electron density contoured at 1σ of A190 jaw residues 1361–1399 (middle and right panel) in crystal form C2-90. Density is also present in crystal form C2-93 (data not shown), whereas it is absent in crystal form C2-100 at the same contour level (left panel). b, Sequence alignment of the Pol I DNA-mimicking loop across different species highlighting the conservation of this element. c, Purified Pol I shows elongation activity in an RNA extension assay. DNA templates (Temp-41 and Temp-27 of 41 and 27 nucleotides, respectively) and 32P-labelled RNA sequences used for the assay are indicated. The autoradiogram shows the elongation of RNA by Pol I producing a run-off of 18 nucleotides (lane 3) or 12 nucleotides (lane 6) depending of the template used. Lanes 1 and 4: the DNA/RNA hybrids were incubated in the absence of Pol I. Lanes 2 and 5: Pol I–DNA–RNA complexes were incubated with a buffer without NTPs. d, Dot spots grown at the indicated temperatures of the parental RPA190 strain and rpa190Δloop strain where the DNA-mimicking loop has been deleted. The rpa190Δloop strain shows a slight temperature-sensitive growth defect on SDC medium.

Extended Data Figure 6 Pol I dimer in the crystal lattice.

The A43 C-terminal tail establishes crystal contacts with a second molecule related by a crystallographic dyad. The A43 C-terminal helix is embedded between the clamp and the protrusion domain of a dyad related molecule. The σA-weighted electron density map (contoured at 1σ) shows clear density corresponding to residues A43 251–326. The two monomers are related by a crystallographic dyad, which is indicated by a dyad symbol.

Extended Data Figure 7 Subunit A12.2 structure and its position in Pol I.

a, Detailed views show the A12.2 Zn sites and the main contacts between its linker and the A190 subunit. The A12.2 linker extends the β-sheet of the A190 jaw. b, The overlap between subunit A12.2 and α-amanitin in the Pol I structure explains the insensitivity of Pol I for this fungal toxin. The Pol II–α-amanitin complex structure (Protein Data Bank accession 2VUM) was superimposed onto the Pol I crystal structure. In the left panel, the α-amanitin toxin is depicted in surface representation (pink). On the right, a detailed view of α-amanitin shows the overlap with the C-terminal Zn ribbon of A12.2.

Extended Data Figure 8 Precise positioning of the A49–A34.5 heterodimer suggests similar positions for the related C37–C53 heterodimer in Pol III and the TFIIF heterodimer in Pol II.

a, Pol I was fitted into the Pol III envelope (EM-1804)12. A49–A34.5 (corresponding to C37–C53 in Pol III), AC40–AC19 and A12.2 (corresponding to C11 in Pol III) are coloured as in Fig. 1. The approximate position of subcomplex C82–C34–C31 is also indicated. b, The proposed Pol II/TFIIF model was manually fitted into the Pol II/TFII-A-B-F/TBP/DNA EM density (EM-2305)43. c, Left panel: detailed view of the anchoring of the A49–A34.5 dimerization domain onto the Pol I core. Right panel: model for the TFIIF dimerization module bound to the Pol II core based on the crystal structures of the human Rap74–Rap30 complex (Protein Data Bank accession 1F3U) and Pol II (Protein Data Bank accession 1WCM).

Extended Data Table 1 Data statistics of the Pol I structure determination
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Fernández-Tornero, C., Moreno-Morcillo, M., Rashid, U. et al. Crystal structure of the 14-subunit RNA polymerase I. Nature 502, 644–649 (2013). https://doi.org/10.1038/nature12636

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