Abstract
The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes. Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA–DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain. This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130° rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation. A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA–DNA hybrid is completely buried in a newly formed, deep protein cavity. Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions. The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
McAllister, W. T. Transcription by T7 RNA polymerase. Nucleic Acids Mol. Biol. 11, 15–25 (1997)
Brieba, L. G. & Sousa, R. T7 promoter release mediated by DNA scrunching. EMBO J. 20, 6826–6835 (2001)
Gunderson, S. I., Chapman, K. A. & Burgess, R. R. Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II). Biochemistry 26, 1539–1546 (1987)
Ikeda, R. A. & Richardson, C. C. Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription. Proc. Natl Acad. Sci. USA 83, 3614–3618 (1986)
Place, C., Oddos, J., Buc, H., McAllister, W. T. & Buckle, M. Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases. Biochemistry 38, 4948–4957 (2000)
Martin, C. T., Muller, D. K. & Coleman, J. E. Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry 27, 3966–3974 (1988)
Liu, C. & Martin, C. T. Promoter clearance by T7 RNA polymerase. J. Biol. Chem. 277, 2725–2731 (2002)
Liu, C. & Martin, C. T. Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase. J. Mol. Biol. 308, 465–475 (2001)
Temiakov, D. et al. The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance. Proc. Natl Acad. Sci. USA 97, 14109–14114 (2000)
Huang, J. & Sousa, R. T7 RNA polymerase elongation complex structure and movement. J. Mol. Biol. 303, 347–358 (2000)
Sousa, R., Chung, Y. J., Rose, J. P. & Wang, B. C. Crystal structure of bacteriophage T7 RNA polymerase at 3.3 Å resolution. Nature 364, 593–599 (1993)
Jeruzalmi, D. & Steitz, T. A. Structure of T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme. EMBO J. 17, 4101–4113 (1998)
Cheetham, G., Jeruzalmi, D. & Steitz, T. A. Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature 399, 80–83 (1999)
Cheetham, G. & Steitz, T. A. Structure of a transcribing T7 RNA polymerase initiation complex. Science 286, 2305–2309 (1999)
Zhang, G. et al. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Å resolution. Cell 98, 811–824 (1999)
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)
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 Ångstrom resolution. Science 292, 1876–1882 (2001)
Murakami, K. S., Musada, S. & Darst, S. A. Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 1280–1284 (2002)
Vassylyev, D. G. et al. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å reslolution. Nature 417, 712–719 (2002)
Cheetham, G. & Steitz, T. A. Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases. Curr. Opin. Struct. Biol. 10, 117–123 (2000)
Severinov, K. T7 RNA polymerase transcription complex: what you see is not what you get. Proc. Natl Acad. Sci. USA 98, 5–7 (2000)
Steitz, T. A. DNA- and RNA-dependent DNA polymerases. Curr. Opin. Struct. Biol. 3, 31–38 (1993)
McAllister, W. T. & Raskin, C. A. The phage RNA polymerases are related to DNA polymerases and reverse transcriptases. Mol. Microbiol. 10, 1–6 (1993)
Temiakov, D., Anikin, M. & McAllister, W. T. Characterization of T7 RNA polymerase transcription complexes assembled on nucleic acid scaffolds. J. Biol. Chem. (in the press)
Temiakov, D. et al. Crystallization and preliminary crystallographic analysis of T7 RNA polymerase elongation complex assembled on an RNA:DNA scaffold. Acta Crystallogr. (submitted)
Kim, Y. et al. Crystal structure of Thermus aquaticus DNA polymerase. Nature 376, 612–616 (1995)
Raskin, C. A., Diaz, G. A. & McAllister, W. T. T7 RNA polymerase mutants with altered promoter specificities. Proc. Natl Acad. Sci. USA 90, 3147–3151 (1993)
Rong, M., He, B., McAllister, W. T. & Durbin, R. K. Promoter specificity determinants of T7 RNA polymerase. Proc. Natl Acad. Sci. USA 95, 515–519 (1998)
Ma, K., Temiakov, D., Jiang, M., Anikin, M. & McAllister, W. T. Major conformational changes occur during the transition from an initiation complex to an elongation complex by T7 RNA polymerase. J. Biol. Chem (in the press)
Mukherjee, S., Brieba, L. G. & Sousa, R. Structural transitions mediating transcription initiation by T7 RNA polymerase. Cell 110, 1–20 (2002)
Mentesana, P. E., Chin-Bow, S. T., Sousa, R. & McAllister, W. T. Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability. J. Mol. Biol. 302, 1049–1062 (2000)
Jiang, M., Rong, M., Martin, C. T. & McAllister, W. T. Interrupting the template strand of the T7 promoter facilitates translocation of the DNA during initiation, reducing transcript slippage and the release of abortive products. J. Mol. Biol. 310, 509–522 (2000)
Bonner, G., Lafer, E. M. & Sousa, R. Characterization of a set of T7 RNA polymerase active site mutants. J. Biol. Chem. 269, 25120–25128 (1994)
Huang, J., Brieba, L. G. & Sousa, R. Misincorporation by wild type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation. Biochemistry 39, 11571–11580 (2000)
Osumi-Davis, P. A., de Aguilera, M. C., Woody, R. W. & Woody, A. Y. Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity. J. Mol. Biol. 226, 37–45 (1992)
Woody, A. Y., Osumi-Davis, P. A., Hiremath, M. M. & Woody, R. W. Pre-steady state and steady-state kinetic studies on transcription initiation catalyed by T7 RNA polymerase and its active site mutants K631R and Y639F. Biochemistry 37, 15958–15964 (1999)
Imburgio, D., Rong, M., Ma, K. & McAllister, W. T. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry 39, 10419–10430 (2000)
He, B., Rong, M., Durbin, R. K. & McAllister, W. T. A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription. J. Mol. Biol. 265, 275–288 (1997)
Brieba, L. G., Gopal, V. & Sousa, R. Scanning mutagenesis reveals roles for helix N of the bacteriophage T7 RNA polymerase thumb subdomain in transcription complex stability, pausing, and termination. J. Biol. Chem. 276, 10306–10313 (2000)
Lyakhov, D. L. et al. Mutant T7 RNA polymerases with altered termination properties. J. Mol. Biol. 269, 28–40 (1997)
Macdonald, L. E., Durbin, R. K., Dunn, J. J. & McAllister, W. T. Characterization of two types of termination signal for bacteriophage T7 RNA polymerase. J. Mol. Biol. 238, 145–158 (1994)
Gopal, V., Brieba, L. G., Guajardo, R., McAllister, W. T. & Sousa, R. Characterization of structural features important for T7 RNAP elongation complex stability reveals competing complex conformations and a role for the non-template strand in RNA displacement. J. Mol. Biol. 290, 411–431 (1999)
Otwinowski, Z. & Minor, W. Processing X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).
Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)
Brunger, A. T. et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)
Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)
Esnouf, R. M. Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. Acta Crystallogr. D 55, 938–940 (1999)
Merrit, E. A. & Bacon, D. J. Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997)
Nichols, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)
Acknowledgements
We thank Y. Kawano for assistance during the data collection at the SPring-8 synchrotron beam line, BL45. We are grateful to A. Murzin for discussions and advice concerning the analysis of the structure. This work was supported in part by grants from the NIH (USA) (W.T.M.) and the Organized Research Combination System of Science and Technology Agency (Japan) (S.Y.).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare that they have no competing financial interests.
Supplementary information
Rights and permissions
About this article
Cite this article
Tahirov, T., Temiakov, D., Anikin, M. et al. Structure of a T7 RNA polymerase elongation complex at 2.9 Å resolution. Nature 420, 43–50 (2002). https://doi.org/10.1038/nature01129
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature01129
This article is cited by
-
Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase
Nature Communications (2023)
-
Observation of structural switch in nascent SAM-VI riboswitch during transcription at single-nucleotide and single-molecule resolution
Nature Communications (2023)
-
Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing
Nature Communications (2022)
-
Identification of a Small Interface between the Methyltransferase and RNA Polymerase of NS5 that is Essential for Zika Virus Replication
Scientific Reports (2018)
-
Structural basis of mitochondrial transcription
Nature Structural & Molecular Biology (2018)
Comments
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.