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


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.

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).


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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).

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Authors and Affiliations



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).

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