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Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein

Nature volume 468, pages 978–982 (2010)Cite this article

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Abstract

The multi-subunit DNA-dependent RNA polymerase (RNAP) is the principal enzyme of transcription for gene expression. Transcription is regulated by various transcription factors. Gre factor homologue 1 (Gfh1), found in the Thermus genus, is a close homologue of the well-conserved bacterial transcription factor GreA, and inhibits transcription initiation and elongation by binding directly to RNAP1,2,3,4,5,6,7,8. The structural basis of transcription inhibition by Gfh1 has remained elusive, although the crystal structures of RNAP and Gfh1 have been determined separately6,7,8,9. Here we report the crystal structure of Thermus thermophilus RNAP complexed with Gfh1. The amino-terminal coiled-coil domain of Gfh1 fully occludes the channel formed between the two central modules of RNAP; this channel would normally be used for nucleotide triphosphate (NTP) entry into the catalytic site. Furthermore, the tip of the coiled-coil domain occupies the NTP β-γ phosphate-binding site. The NTP-entry channel is expanded, because the central modules are ‘ratcheted’ relative to each other by ∼7°, as compared with the previously reported elongation complexes. This ‘ratcheted state’ is an alternative structural state, defined by a newly acquired contact between the central modules. Therefore, the shape of Gfh1 is appropriate to maintain RNAP in the ratcheted state. Simultaneously, the ratcheting expands the nucleic-acid-binding channel, and kinks the bridge helix, which connects the central modules. Taken together, the present results reveal that Gfh1 inhibits transcription by preventing NTP binding and freezing RNAP in the alternative structural state. The ratcheted state might also be associated with other aspects of transcription, such as RNAP translocation and transcription termination.

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Figure 1: Structure of EC·Gfh1.
Figure 2: Ratcheting of the shelf module.
Figure 3: The bridge helix and Gfh1.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structures of EC·Gfh1 have been deposited in the Protein Data Bank, under accession numbers 3AOH (crystal 1) and 3AOI (crystal 2).

References

  1. Borukhov, S., Polyakov, A., Nikiforov, V. & Goldfarb, A. GreA protein: a transcription elongation factor from Escherichia coli . Proc. Natl Acad. Sci. USA 89, 8899–8902 (1992)

    Article  CAS  ADS  Google Scholar 

  2. Stebbins, C. E. et al. Crystal structure of the GreA transcript cleavage factor from Escherichia coli . Nature 373, 636–640 (1995)

    Article  CAS  ADS  Google Scholar 

  3. Vassylyeva, M. N. et al. The carboxy-terminal coiled-coil of the RNA polymerase β'-subunit is the main binding site for Gre factors. EMBO Rep. 8, 1038–1043 (2007)

    Article  CAS  Google Scholar 

  4. Hogan, B. P., Hartsch, T. & Erie, D. A. Transcript cleavage by Thermus thermophilus RNA polymerase. Effects of GreA and anti-GreA factors. J. Biol. Chem. 277, 967–975 (2002)

    Article  CAS  Google Scholar 

  5. Laptenko, O. & Borukhov, S. Biochemical assays of Gre factors of Thermus thermophilus . Methods Enzymol. 371, 219–232 (2003)

    Article  CAS  Google Scholar 

  6. Lamour, V., Hogan, B. P., Erie, D. A. & Darst, S. A. Crystal structure of Thermus aquaticus Gfh1, a Gre-factor paralog that inhibits rather than stimulates transcript cleavage. J. Mol. Biol. 356, 179–188 (2006)

    Article  CAS  Google Scholar 

  7. Laptenko, O. et al. pH-dependent conformational switch activates the inhibitor of transcription elongation. EMBO J. 25, 2131–2141 (2006)

    Article  CAS  Google Scholar 

  8. Symersky, J. et al. Regulation through the RNA polymerase secondary channel. Structural and functional variability of the coiled-coil transcription factors. J. Biol. Chem. 281, 1309–1312 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  12. 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  CAS  ADS  Google Scholar 

  13. Vassylyev, D. G., Vassylyeva, M. N., Perederina, A., Tahirov, T. H. & Artsimovitch, I. Structural basis for transcription elongation by bacterial RNA polymerase. Nature 448, 157–162 (2007)

    Article  CAS  ADS  Google Scholar 

  14. Vassylyev, D. G. et al. Structural basis for substrate loading in bacterial RNA polymerase. Nature 448, 163–168 (2007)

    Article  CAS  ADS  Google Scholar 

  15. Wang, D., Bushnell, D. A., Westover, K. D., Kaplan, C. D. & Kornberg, R. D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127, 941–954 (2006)

    Article  CAS  Google Scholar 

  16. Westover, K. D., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II. Science 303, 1014–1016 (2004)

    Article  CAS  ADS  Google Scholar 

  17. Westover, K. D., Bushnell, D. A. & Kornberg, R. D. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center. Cell 119, 481–489 (2004)

    Article  CAS  Google Scholar 

  18. 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  Google Scholar 

  19. Brueckner, F. & Cramer, P. Structural basis of transcription inhibition by α-amanitin and implications for RNA polymerase II translocation. Nature Struct. Mol. Biol. 15, 811–818 (2008)

    Article  CAS  Google Scholar 

  20. Sydow, J. F. et al. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol. Cell 34, 710–721 (2009)

    Article  CAS  Google Scholar 

  21. Wang, D. et al. Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution. Science 324, 1203–1206 (2009)

    Article  CAS  ADS  Google Scholar 

  22. Temiakov, D. et al. Structural basis of transcription inhibition by antibiotic streptolydigin. Mol. Cell 19, 655–666 (2005)

    Article  CAS  Google Scholar 

  23. Tuske, S. et al. Inhibition of bacterial RNA polymerase by streptolydigin: stabilization of a straight-bridge-helix active-center conformation. Cell 122, 541–552 (2005)

    Article  CAS  Google Scholar 

  24. Tagami, S., Sekine, S., Kumarevel, T., Yamamoto, M. & Yokoyama, S. Crystallization and preliminary X-ray crystallographic analysis of Thermus thermophilus transcription elongation complex bound to Gfh1. Acta Crystallogr. F 66, 64–68 (2010)

    Article  CAS  ADS  Google Scholar 

  25. Darst, S. A. et al. Conformational flexibility of bacterial RNA polymerase. Proc. Natl Acad. Sci. USA 99, 4296–4301 (2002)

    Article  CAS  ADS  Google Scholar 

  26. Murakami, K. S., Masuda, S. & Darst, S. A. Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 1280–1284 (2002)

    Article  CAS  ADS  Google Scholar 

  27. 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  Google Scholar 

  28. 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  Google Scholar 

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

    Article  Google Scholar 

  30. Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

    Article  CAS  Google Scholar 

  31. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  34. Mukhopadhyay, J. et al. The RNA polymerase “switch region” is a target for inhibitors. Cell 135, 295–307 (2008)

    Article  CAS  Google Scholar 

  35. Abyzov, A., Bjornson, R., Felipe, M. & Gerstein, M. RigidFinder: a fast and sensitive method to detect rigid blocks in large macromolecular complexes. Proteins 78, 309–324 (2010)

    Article  CAS  Google Scholar 

  36. Vassylyeva, M. N. et al. Purification, crystallization and initial crystallographic analysis of RNA polymerase holoenzyme from Thermus thermophilus . Acta Crystallogr. D 58, 1497–1500 (2002)

    Article  Google Scholar 

  37. Kashkina, E. et al. Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations. Nucleic Acids Res. 34, 4036–4045 (2006)

    Article  CAS  Google Scholar 

  38. Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli . Proc. Natl Acad. Sci. USA 99, 11020–11024 (2002)

    Article  CAS  ADS  Google Scholar 

  39. Sakamoto, K. et al. Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography. Structure 17, 335–344 (2009)

    Article  CAS  Google Scholar 

  40. Chumpolkulwong, N. et al. Translation of 'rare' codons in a cell-free protein synthesis system from Escherichia coli . J. Struct. Funct. Genomics 7, 31–36 (2006)

    Article  CAS  Google Scholar 

  41. Hino, N. et al. Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nature Methods 2, 201–206 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is based on experiments performed at SPring-8 (with the approval of the Japan Synchrotron Radiation Research Institute) and at the Swiss Light Source (SLS). We thank N. Shimizu for supporting our data collection at SPring-8 beamline BL41XU; T. Tomizaki and C. Schulze-Briese for supporting our data collection at SLS beamline X06SA; and Y. Fujii for assisting with our data collection and for comments. We thank T. Tanaka and K. Sakamoto for assistance in protein preparation. This work was supported in part by a Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (to S.-i.S.), a JSPS Grant-in-Aid for Scientific Research (to S.i.-S. and S.Y.), and the Targeted Proteins Research Program (TPRP), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. S.T. was supported by the JSPS Global Centers of Excellence Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms).

Author information

Author notes

  1. Thirumananseri Kumarevel

    Present address: Present address: Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.,

Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Shunsuke Tagami, Shun-ichi Sekine, Yuko Murayama, Syunsuke Kamegamori & Shigeyuki Yokoyama

  2. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, 230-0045, Yokohama, Japan

    Shunsuke Tagami, Shun-ichi Sekine, Nobumasa Hino, Yuko Murayama, Syunsuke Kamegamori, Kensaku Sakamoto & Shigeyuki Yokoyama

  3. Laboratory of Structural Biology, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Shun-ichi Sekine & Shigeyuki Yokoyama

  4. Structural and Molecular Biology Laboratory, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, 679-5148, Hyogo, Japan

    Thirumananseri Kumarevel

  5. SR Life Science Instrumentation Unit, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, 679-5148, Hyogo, Japan

    Masaki Yamamoto

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Contributions

S.T., S.-i.S., T. K. and S.Y. designed the research. S.T. and S.-i.S. performed the structural analysis. M.Y. supported the structural analysis. S.T., S.-i.S., N.H., S.K. and K.S. performed the disulphide-bonding and/or photo-crosslinking analyses. S.T. and Y.M. performed the biochemical analysis of Gre factors. S.-i.S. created the movies. S.T., S.-i.S. and S.Y. wrote the paper.

Corresponding authors

Correspondence to Shun-ichi Sekine or Shigeyuki Yokoyama.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2, Supplementary Text 1-17, Supplementary Figures 1-27 with legends, Full legends for Supplementary Movies 1-2 and additional references. (PDF 9334 kb)

Supplementary Movie 1

This movie shows the present EC·Gfh1 in the ‘ratcheted’ state and its differences from the ‘tight’ state seen in the previous ECs (see Supplementary information file page 52 for full legend). (MOV 18974 kb)

Supplementary Movie 2

This movie shows the difference in the clamp module orientation between EC·Gfh1 and the previous EC (PDB 2O5I) (see Supplementary information file page 53 for full legend). (MOV 3665 kb)

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Tagami, S., Sekine, Si., Kumarevel, T. et al. Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein. Nature 468, 978–982 (2010). https://doi.org/10.1038/nature09573

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