Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structure of the 14-subunit RNA polymerase I

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

Subjects

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.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

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.

References

  1. Warner, J. R. The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24, 437–440 (1999)

    Article  CAS  PubMed  Google Scholar 

  2. Drygin, D., Rice, W. G. & Grummt, I. The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer. Annu. Rev. Pharmacol. Toxicol. 50, 131–156 (2010)

    Article  CAS  PubMed  Google Scholar 

  3. Grummt, I. Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev. 17, 1691–1702 (2003)

    Article  CAS  PubMed  Google Scholar 

  4. Moss, T., Langlois, F., Gagnon-Kugler, T. & Stefanovsky, V. A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell. Mol. Life Sci. 64, 29–49 (2007)

    Article  CAS  PubMed  Google Scholar 

  5. Vannini, A. & Cramer, P. Conservation between the RNA polymerase I, II, and III transcription initiation machineries. Mol. Cell 45, 439–446 (2012)

    Article  CAS  PubMed  Google Scholar 

  6. Russell, J. & Zomerdijk, J. C. The RNA polymerase I transcription machinery. Biochem. Soc. Symp. 73, 203–216 (2006)

    Article  CAS  Google Scholar 

  7. Kuhn, C. D. et al. Functional architecture of RNA polymerase I. Cell 131, 1260–1272 (2007)

    Article  CAS  PubMed  Google Scholar 

  8. Ruan, W., Lehmann, E., Thomm, M., Kostrewa, D. & Cramer, P. Evolution of two modes of intrinsic RNA polymerase transcript cleavage. J. Biol. Chem. 286, 18701–18707 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Carter, R. & Drouin, G. The increase in the number of subunits in eukaryotic RNA polymerase III relative to RNA polymerase II is due to the permanent recruitment of general transcription factors. Mol. Biol. Evol. 27, 1035–1043 (2010)

    Article  CAS  PubMed  Google Scholar 

  10. Geiger, S. R. et al. RNA polymerase I contains a TFIIF-related DNA-binding subcomplex. Mol. Cell 39, 583–594 (2010)

    Article  CAS  PubMed  Google Scholar 

  11. Cramer, P. et al. Structure of eukaryotic RNA polymerases. Ann. Rev. Biophys. 37, 337–352 (2008)

    Article  CAS  Google Scholar 

  12. Fernández-Tornero, C. et al. Conformational flexibility of RNA polymerase III during transcriptional elongation. EMBO J. 29, 3762–3772 (2010)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Bischler, N. et al. Localization of the yeast RNA polymerase I-specific subunits. EMBO J. 21, 4136–4144 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Schultz, P., Celia, H., Riva, M., Sentenac, A. & Oudet, P. Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals. EMBO J. 12, 2601–2607 (1993)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jennebach, S., Herzog, F., Aebersold, R. & Cramer, P. Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage. Nucleic Acids Res. 40, 5591–5601 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Fernández-Tornero, C. et al. Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III. Mol. Cell 25, 813–823 (2007)

    Article  PubMed  CAS  Google Scholar 

  17. Cramer, P., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: RNA polymerase II at 2.8 Ångstrom resolution. Science 292, 1863–1876 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Armache, K. J., Kettenberger, H. & Cramer, P. Architecture of initiation-competent 12-subunit RNA polymerase II. Proc. Natl Acad. Sci. USA 100, 6964–6968 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bushnell, D. A. & Kornberg, R. D. Complete, 12-subunit RNA polymerase II at 4.1-Å resolution: implications for the initiation of transcription. Proc. Natl Acad. Sci. USA 100, 6969–6973 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gnatt, A. L., Cramer, P., Fu, J., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 Å resolution. Science 292, 1876–1882 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Kettenberger, H., Armache, K. J. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004)

    Article  CAS  PubMed  Google Scholar 

  23. Hirata, A., Klein, B. J. & Murakami, K. S. The X-ray crystal structure of RNA polymerase from Archaea. Nature 451, 851–854 (2008)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang, G. et al. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell 98, 811–824 (1999)

    Article  CAS  PubMed  Google Scholar 

  25. Vassylyev, D. G. et al. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417, 712–719 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Tan, L., Wiesler, S., Trzaska, D., Carney, H. C. & Weinzierl, R. O. Bridge helix and trigger loop perturbations generate superactive RNA polymerases. J. Biol. 7, 40 (2008)

    Article  PubMed  PubMed Central  Google Scholar 

  27. Jovanovic, M. et al. Activity map of the Escherichia coli RNA polymerase bridge helix. J. Biol. Chem. 286, 14469–14479 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Weinzierl, R. O. The bridge helix of RNA polymerase acts as a central nanomechanical switchboard for coordinating catalysis and substrate movement. Archaea 2011, 608385 (2011)

    Article  PubMed  CAS  Google Scholar 

  29. Edwards, A. M., Kane, C. M., Young, R. A. & Kornberg, R. D. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro . J. Biol. Chem. 266, 71–75 (1991)

    Article  CAS  PubMed  Google Scholar 

  30. Mosley, A. L. et al. Quantitative proteomics demonstrates that the RNA polymerase II subunits Rpb4 and Rpb7 dissociate during transcription elongation. Mol. Cell. Proteomics 12, 1230–1538 (2013)

    Article  CAS  Google Scholar 

  31. Huet, J., Buhler, J. M., Sentenac, A. & Fromageot, P. Dissociation of two polypeptide chains from yeast RNA polymerase A. Proc. Natl Acad. Sci. USA 72, 3034–3038 (1975)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Peyroche, G. et al. The recruitment of RNA polymerase I on rDNA is mediated by the interaction of the A43 subunit with Rrn3. EMBO J. 19, 5473–5482 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Blattner, C. et al. Molecular basis of Rrn3-regulated RNA polymerase I initiation and cell growth. Genes Dev. 25, 2093–2105 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Milkereit, P., Schultz, P. & Tschochner, H. Resolution of RNA polymerase I into dimers and monomers and their function in transcription. Biol. Chem. 378, 1433–1443 (1997)

    Article  CAS  PubMed  Google Scholar 

  35. Kettenberger, H., Armache, K. J. & Cramer, P. Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 114, 347–357 (2003)

    Article  CAS  PubMed  Google Scholar 

  36. Jeon, C., Yoon, H. & Agarwal, K. The transcription factor TFIIS zinc ribbon dipeptide Asp-Glu is critical for stimulation of elongation and RNA cleavage by RNA polymerase II. Proc. Natl Acad. Sci. USA 91, 9106–9110 (1994)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  37. Prescott, E. M. et al. Transcriptional termination by RNA polymerase I requires the small subunit Rpa12p. Proc. Natl Acad. Sci. USA 101, 6068–6073 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kedinger, C., Gniazdowski, M., Mandel, J. L., Jr, Gissinger, F. & Chambon, P. α-Amanitin: a specific inhibitor of one of two DNA-pendent RNA polymerase activities from calf thymus. Biochem. Biophys. Res. Commun. 38, 165–171 (1970)

    Article  CAS  PubMed  Google Scholar 

  39. Weinmann, R. & Roeder, R. G. Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes. Proc. Natl Acad. Sci. USA 71, 1790–1794 (1974)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. De Carlo, S., Carles, C., Riva, M. & Schultz, P. Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I. J. Mol. Biol. 329, 891–902 (2003)

    Article  CAS  PubMed  Google Scholar 

  41. Chen, Z. A. et al. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29, 717–726 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Eichner, J., Chen, H. T., Warfield, L. & Hahn, S. Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex. EMBO J. 29, 706–716 (2010)

    Article  CAS  PubMed  Google Scholar 

  43. He, Y., Fang, J., Taatjes, D. J. & Nogales, E. Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Beckouet, F. et al. Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription. Mol. Cell. Biol. 28, 1596–1605 (2008)

    Article  CAS  PubMed  Google Scholar 

  45. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bricogne, G., Vonrhein, C., Flensburg, C., Schiltz, M. & Paciorek, W. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

    Article  CAS  PubMed  Google Scholar 

  48. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  PubMed  Google Scholar 

  49. Skubák, P., Murshudov, G. N. & Pannu, N. S. Direct incorporation of experimental phase information in model refinement. Acta Crystallogr. D 60, 2196–2201 (2004)

    Article  PubMed  CAS  Google Scholar 

  50. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  PubMed  Google Scholar 

  53. Wittekind, M. et al. Isolation and characterization of temperature-sensitive mutations in RPA190, the gene encoding the largest subunit of RNA polymerase I from Saccharomyces cerevisiae . Mol. Cell. Biol. 8, 3997–4008 (1988)

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004)

    Article  CAS  PubMed  Google Scholar 

  55. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors
  1. Carlos Fernández-Tornero
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. María Moreno-Morcillo
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Umar J. Rashid
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Nicholas M. I. Taylor
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Federico M. Ruiz
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Tim Gruene
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Pierre Legrand
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Ulrich Steuerwald
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Christoph W. Müller
    View author publications

    You can also search for this author inPubMed Google Scholar

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.

Corresponding authors

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

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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
Full size table

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature12636

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing