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Structural basis for initiation of transcription from an RNA polymerase–promoter complex

Nature volume 399, pages 80–83 (1999)Cite this article

A Correction to this article was published on 01 July 1999

Abstract

Although the single-polypeptide-chain RNA polymerase from bacteriophage T7 (T7RNAP), like other RNA polymerases, uses the same mechanism of polymerization as the DNA polymerases, it can also recognize a specific promoter sequence, initiate new RNA chains from a single nucleotide, abortively cycle the synthesis of short transcripts, be regulated by a transcription inhibitor, and terminate transcription1,2,3. As T7RNAP is homologous to the Pol I family of DNA polymerases4, the differences between the structure of T7RNAP complexed to substrates and that of the corresponding DNA polymerase complex provides a structural basis for understanding many of these functional differences. T7RNAP initiates RNA synthesis at promoter sequences that are conserved from positions −17 to +6 relative to the start site of transcription. The crystal structure at 2.4 Å resolution of T7RNAP complexed with a 17-base-pair promoter shows that the four base pairs closest to the catalytic active site have melted to form a transcription bubble. The T7 promoter sequence is recognized by interactions in the major groove between an antiparallel β-loop and bases. The amino-terminal domain is involved in promoter recognition and DNA melting. We have also used homology modelling of the priming and incoming nucleoside triphosphates from the T7 DNA-polymerase ternary complex structure to explain the specificity of T7RNAP for ribonucleotides, its ability to initiate from a single nucleotide, and the abortive cycling at the initiation of transcription.

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Figure 1: Surface representation of the T7RNAP–promoter complex structure.
Figure 2: Interactions between the T7-promoter DNA and three regions of T7RNAP that are unique to the RNA polymerase.
Figure 3: Homology-modelled initiation complex.

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References

  1. Steitz, T. A., Smerdon, S. J., Jaeger, J. & Joyce, C. M. Aunified polymerase mechanism for nonhomologous DNA and RNA polymerase. Science 266, 2022–2025 (1994).

    Article  ADS  CAS  Google Scholar 

  2. Carpousis, A. J. & Gralla, J. D. Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry 19, 3245–3253 (1980).

    Article  CAS  Google Scholar 

  3. McAllister, W. T. & Wu, H.-L. Regulation of transcription of the late genes of bacteriophage T7. Proc. Natl Acad. Sci. USA 75, 804–808 (1978).

    Article  ADS  CAS  Google Scholar 

  4. Delarue, M., Poch, O., Tordo, N., Moras, D. & Argos, P. An attempt to unify the structure of polymerases. Protein Eng. 3, 461–467 (1990).

    Article  CAS  Google Scholar 

  5. Raskin, C. A., Diaz, G., Joho, K. & McAllister, W. T. Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity. J. Mol. Biol. 228, 506–515 (1992).

    Article  CAS  Google Scholar 

  6. Lee, S. S. & Kang, C. Atwo base-pair substitution in T7 promoters by SP6 promoter-specific base-pairs alone abolishes T7 promoter activity but reveals SP6 promoter activity. Biochem. Int. 26, 1–5 (1992).

    CAS  PubMed  Google Scholar 

  7. Masters, B. S., Stohl, L. L. & Clayton, D. A. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophage T3 and T7. Cell 51, 89–99 (1987).

    Article  CAS  Google Scholar 

  8. 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).

    Article  ADS  CAS  Google Scholar 

  9. Jeruzalmi, D. & Steitz, T. A. Structure of the T7 RNA polymerase complexed to the transcriptional inhibitor T7 lysozyme. EMBO J. 17, 4101–4113 (1998).

    Article  CAS  Google Scholar 

  10. Souza, R., Chung, Y. J., Rose, J. P. & Wang, B.-C. Crystal structure of bacteriophage T7 RNA polymerase at 3.3 Å resolution. Nature 364, 595–599 (1993).

    ADS  Google Scholar 

  11. Steitz, T. A. DNA- and RNA-dependent DNA polymerases. Curr. Opin. Struct. Biol. 3, 31–38 (1993).

    Article  CAS  Google Scholar 

  12. Eom, S. H., Wang, J. & Steitz, T. A. Structure of Taq polymerase with DNA at the polymerase active site. Nature 382, 278–281 (1996).

    Article  ADS  CAS  Google Scholar 

  13. Doublié, S., Tabor, S., Long, A. M., Richardson, C. C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258 (1998).

    Article  ADS  Google Scholar 

  14. Steitz, T. A. Structural studies of protein–nucleic acid interactions: The sources of sequence specific binding. Q. Rev. Biophys. 23, 205–280 (1990).

    Article  CAS  Google Scholar 

  15. Rice, P. A., Yang, S., Mizuuchi, K. & Nash, H. A. Crystal structure of an IHF–DNA complex: A protein induced DNA U-turn. Cell 87, 1295–1306 (1996).

    Article  CAS  Google Scholar 

  16. Somers, W. S. & Phillips, S. E. V. Crystal structure of the met repressor–operator complex at 2.8 Å resolution reveals DNA recognition by β-strands. Nature 359, 387–393 (1992).

    Article  ADS  CAS  Google Scholar 

  17. Raumann, B. E., Rould, M. A., Pabo, C. O. & Sauer, R. T. DNA recognition by beta-sheets in the Arc repressor-operator crystal structure. Nature 367, 754–757 (1994).

    Article  ADS  CAS  Google Scholar 

  18. Suzuki, M. DNA recognition by a β-sheet. Protein Eng. 8, 1–4 (1995).

    Article  CAS  Google Scholar 

  19. Kim, Y., Geiger, J. H., Hahn, S. & Sigler, P. B. Crystal structure of a yeast TBP/TATA-box complex. Nature 365, 512–520 (1993).

    Article  ADS  CAS  Google Scholar 

  20. Kim, J. L., Nikolov, D. B. & Burley, S. K. Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature 365, 520–527 (1993).

    Article  ADS  CAS  Google Scholar 

  21. Rould, M. A. & Steitz, T. A. Structure of the glutaminyl-tRNA synthetase-tRNAGlu-ATP complex. Nucleic Acids Mol. Biol. 6, 225–245 (1992).

    Article  CAS  Google Scholar 

  22. Osumi-Davis, P. A., Aguilera, M. C., Woody, R. W. & Woody, A. Y. M. Asp 357, Asp 812 are essential and Lys 631, His 811 are catalytically significant in bacteriophage T7 RNA polymerase activity. J. Mol. Biol. 226, 37–45 (1992).

    Article  CAS  Google Scholar 

  23. Gao, G., Orlova, M., Georgiadis, M. M., Hendrickson, W. A. & Goff, S. P. Conferring RNA polymerase activity to a DNA polymerase: A single residue in reverse transcriptase controls substrate selection. Proc. Natl Acad. Sci. USA 94, 407–411 (1997).

    Article  ADS  CAS  Google Scholar 

  24. Davanloo, P., Rosenberg, A. H., Dunn, J. J. & Studier, F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl Acad. Sci. USA 81, 2035–2039 (1984).

    Article  ADS  CAS  Google Scholar 

  25. Leslie, A. G. W. Joint CCP4 and ESF-EACMB Newsletter Protein crystallogr. No. 26 (Daresbury Laboratory, Warrington, UK, (1992).

  26. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  27. Collaborative Computational Project No. 4. The CCP4 Suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  28. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 52, 30–42 (1996).

    Article  CAS  Google Scholar 

  29. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

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

    Article  Google Scholar 

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Acknowledgements

We thank many members of the T.A.S. laboratory for assistance with data collection at beamlines X12C (NSLS, Brookhaven Laboratory), A1, F1 and F2 (Cornell High Energy Synchrotron). This work was supported by a grant from the N.I.H.

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Author notes

  1. David Jeruzalmi

    Present address: Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, New York, 10021, USA

Authors and Affiliations

  1. Departments of Molecular Biophysics and Biochemistry, 266 Whitney Avenue, New Haven, 06520-8114, Connecticut, USA

    Graham M. T. Cheetham, David Jeruzalmi & Thomas A Steitz

  2. Departments of Chemistry, 266 Whitney Avenue, New Haven, 06520-8114, Connecticut, USA

    Thomas A Steitz

  3. Howard Hughes Medical Institute, Yale University, 266 Whitney Avenue, New Haven, 06520-8114, Connecticut, USA

    Graham M. T. Cheetham & Thomas A Steitz

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  2. David Jeruzalmi
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Cheetham, G., Jeruzalmi, D. & Steitz, T. Structural basis for initiation of transcription from an RNA polymerase–promoter complex. Nature 399, 80–83 (1999). https://doi.org/10.1038/19999

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