Article | Published:

Structural basis of RNA polymerase III transcription initiation

Nature volume 553, pages 301306 (18 January 2018) | Download Citation


RNA polymerase (Pol) III transcribes essential non-coding RNAs, including the entire pool of transfer RNAs, the 5S ribosomal RNA and the U6 spliceosomal RNA, and is often deregulated in cancer cells. The initiation of gene transcription by Pol III requires the activity of the transcription factor TFIIIB to form a transcriptionally active Pol III preinitiation complex (PIC). Here we present electron microscopy reconstructions of Pol III PICs at 3.4–4.0 Å and a reconstruction of unbound apo-Pol III at 3.1 Å. TFIIIB fully encircles the DNA and restructures Pol III. In particular, binding of the TFIIIB subunit Bdp1 rearranges the Pol III-specific subunits C37 and C34, thereby promoting DNA opening. The unwound DNA directly contacts both sides of the Pol III cleft. Topologically, the Pol III PIC resembles the Pol II PIC, whereas the Pol I PIC is more divergent. The structures presented unravel the molecular mechanisms underlying the first steps of Pol III transcription and also the general conserved mechanisms of gene transcription initiation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from $8.99

All prices are NET prices.



  1. 1.

    et al. RNA polymerase III limits longevity downstream of TORC1. Nature (2017)

  2. 2.

    et al. Redox signaling by the RNA polymerase III TFIIB-related factor Brf2. Cell 163, 1375–1387 (2015)

  3. 3.

    et al. Modulated expression of specific tRNAs drives gene expression and cancer progression. Cell 165, 1416–1427 (2016)

  4. 4.

    et al. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome. Nat. Genet. 43, 20–22 (2011)

  5. 5.

    et al. Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III. Nat. Commun. 6, 7623 (2015)

  6. 6.

    , & Differential phosphorylation of RNA polymerase III and the initiation factor TFIIIB in Saccharomyces cerevisiae. PLoS ONE 10, e0127225 (2015)

  7. 7.

    et al. Direct regulation of tRNA and 5S rRNA gene transcription by Polo-like kinase 1. Mol. Cell 45, 541–552 (2012)

  8. 8.

    et al. Molecular basis of RNA polymerase III transcription repression by Maf1. Cell 143, 59–70 (2010)

  9. 9.

    et al. Genome-wide location of yeast RNA polymerase III transcription machinery. EMBO J. 22, 4738–4747 (2003)

  10. 10.

    et al. Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells. Nat. Struct. Mol. Biol. 17, 635–640 (2010)

  11. 11.

    et al. Pol II and its associated epigenetic marks are present at Pol III-transcribed noncoding RNA genes. Nat. Struct. Mol. Biol. 17, 629–634 (2010)

  12. 12.

    , & A minimal RNA polymerase III transcription system. EMBO J. 18, 5042–5051 (1999)

  13. 13.

    , , & S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors. Cell 60, 235–245 (1990)

  14. 14.

    , , , & TFIIIC-independent in vitro transcription of yeast tRNA genes. J. Mol. Biol. 299, 601–613 (2000)

  15. 15.

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

  16. 16.

    , & Essential roles of Bdp1, a subunit of RNA polymerase III initiation factor TFIIIB, in transcription and tRNA processing. Mol. Cell. Biol. 22, 3264–3275 (2002)

  17. 17.

    , & Dual role of the C34 subunit of RNA polymerase III in transcription initiation. EMBO J. 16, 5730–5741 (1997)

  18. 18.

    , & The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening. J. Biol. Chem. 285, 2695–2706 (2010)

  19. 19.

    & Mechanism of transcription termination by RNA polymerase III utilizes a non-template strand sequence-specific signal element. Mol. Cell 58, 1124–1132 (2015)

  20. 20.

    , , & Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB. Proc. Natl Acad. Sci. USA 102, 15406–15411 (2005)

  21. 21.

    , & The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening. EMBO J. 20, 2823–2834 (2001)

  22. 22.

    et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016)

  23. 23.

    et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016)

  24. 24.

    , , & Formation of open and elongating transcription complexes by RNA polymerase III. J. Mol. Biol. 226, 47–58 (1992)

  25. 25.

    , , , & Crystal structure of a transcription factor IIIB core interface ternary complex. Nature 422, 534–539 (2003)

  26. 26.

    , , & A cryptic DNA binding domain at the COOH terminus of TFIIIB70 affects formation, stability, and function of preinitiation complexes. J. Biol. Chem. 272, 18341–18349 (1997)

  27. 27.

    et al. Molecular mechanisms of Bdp1 in TFIIIB assembly and RNA polymerase III transcription initiation. Nat. Commun. 8, 130 (2017)

  28. 28.

    et al. Molecular structures of unbound and transcribing RNA polymerase III. Nature 528, 231–236 (2015)

  29. 29.

    , , & A region of Bdp1 necessary for transcription initiation that is located within the RNA polymerase III active site cleft. Mol. Cell. Biol. 35, 2831–2840 (2015)

  30. 30.

    , & The TFIIF-like Rpc37/53 dimer lies at the center of a protein network to connect TFIIIC, Bdp1, and the RNA polymerase III active center. Mol. Cell. Biol. 31, 2715–2728 (2011)

  31. 31.

    , , , & Kinetic trapping of DNA by transcription factor IIIB. Proc. Natl Acad. Sci. USA 98, 9581–9586 (2001)

  32. 32.

    , & Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III. Mol. Cell. Biol. 15, 1467–1478 (1995)

  33. 33.

    & RNA polymerase III mutants in TFIIFα-like C37 that cause terminator readthrough with no decrease in transcription output. Nucleic Acids Res. 41, 139–155 (2013)

  34. 34.

    , , & Structural basis of transcriptional pausing in bacteria. Cell 152, 431–441 (2013)

  35. 35.

    et al. Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein. Nature 468, 978–982 (2010)

  36. 36.

    , , & Structural visualization of key steps in human transcription initiation. Nature 495, 481–486 (2013)

  37. 37.

    et al. TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle. Proc. Natl Acad. Sci. USA 113, E1816–E1825 (2016)

  38. 38.

    et al. The X-ray crystal structure of the euryarchaeal RNA polymerase in an open-clamp configuration. Nat. Commun. 5, 5132 (2014)

  39. 39.

    et al. Structural basis of RNA polymerase I transcription initiation. Cell 169, 120–131 (2017)

  40. 40.

    et al. Structural mechanism of ATP-independent transcription initiation by RNA polymerase I. eLife 6, e27414 (2017)

  41. 41.

    et al. Structural insights into transcription initiation by yeast RNA polymerase I. EMBO J. 36, 2698–2709 (2017)

  42. 42.

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

  43. 43.

    & 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)

  44. 44.

    et al. Opening and closing of the bacterial RNA polymerase clamp. Science 337, 591–595 (2012)

  45. 45.

    et al. Structural basis of transcription initiation. Science 338, 1076–1080 (2012)

  46. 46.

    & Events during initiation of archaeal transcription: open complex formation and DNA-protein interactions. J. Bacteriol. 183, 3025–3031 (2001)

  47. 47.

    et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017)

  48. 48.

    & CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

  49. 49.

    , , & Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016)

  50. 50.

    , , & cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017)

  51. 51.

    I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008)

  52. 52.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  53. 53.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

  54. 54.

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

  55. 55.

    et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  56. 56.

    , & Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)

Download references


We thank N. Cronin at the Institute of Cancer Research for help with yeast fermentation; C. Richardson for the computing infrastructure; C. Plaschka (MRC-LMB, Cambridge) for advice during data processing; and the staff at beamline M03 of the Diamond Light Source synchrotron (UK) for help with EM data collection (EM15629, EM16599 and EM166601). We acknowledge support and the use of resources of iNEXT, in particular C. Sachse and W. Hagen for EM data collection at EMBL Heidelberg (PID1956 and 2180). G.A.-P. is a recipient of a Marie Sklodowska-Curie Intra-European Fellowship (EU project 655238). E.M. is supported by Cancer Research UK (CR-UK C12209/A16749). A.V. is supported by a Biotechnology and Biological Sciences Research Council (BBSRC) New Investigator Award (BB/K014390/1), a Cancer Research UK Programme Foundation (CR-UK C47547/A21536) and a Wellcome Trust Investigator Award (200818/Z/16/Z).

Author information


  1. The Institute of Cancer Research, London SW7 3RP, UK

    • Guillermo Abascal-Palacios
    • , Ewan Phillip Ramsay
    • , Fabienne Beuron
    • , Edward Morris
    •  & Alessandro Vannini


  1. Search for Guillermo Abascal-Palacios in:

  2. Search for Ewan Phillip Ramsay in:

  3. Search for Fabienne Beuron in:

  4. Search for Edward Morris in:

  5. Search for Alessandro Vannini in:


G.A.-P. carried out yeast fermentation, Pol III purification, Pol III PIC reconstitution, EM specimen preparation, EM data collection and processing, model building and refinement. E.P.R. carried out EM data collection and processing of the Bdp1Δ(355–372) Pol III PIC and helped with EM data processing and analysis. F.B. carried out cryo-EM sample preparation, screening and sample collection. E.M. helped during initial EM characterization and data collection. A.V. designed and supervised research, analysed the structural data and prepared the manuscript with contributions from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alessandro Vannini.

Reviewer Information Nature thanks R. Maraia and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary


  1. 1.

    Conformational changes upon PIC and open complex formation

    Morphing between cPol3 and OC-PIC cryo-EM structures shows the structural rearrangements occurring during PIC formation, which result in the lock of the Pol III clamp and the stabilisation of the downstream edge of the DNA bubble. The morphing and the video were generated using Chimera55. OC-PIC core subunits are depicted as grey molecular surfaces. The C82/C34/C31 subcomplex, stalk, TFIIIB subunits and C160 clamp helices are shown as ribbon and coloured as in Figure 1b.

About this article

Publication history






By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.