Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecular basis for RNA polymerization by Qβ replicase

Nature Structural & Molecular Biology volume 19, pages 229–237 (2012)Cite this article

Subjects

Abstract

Core Qβ replicase comprises the Qβ virus–encoded RNA-dependent RNA polymerase (β-subunit) and the host Escherichia coli translational elongation factors EF-Tu and EF-Ts. The functions of the host proteins in the viral replicase are not clear. Structural analyses of RNA polymerization by core Qβ replicase reveal that at the initiation stage, the 3′-adenine of the template RNA provides a stable platform for de novo initiation. EF-Tu in Qβ replicase forms a template exit channel with the β-subunit. At the elongation stages, the C-terminal region of the β-subunit, assisted by EF-Tu, splits the temporarily double-stranded RNA between the template and nascent RNAs before translocation of the single-stranded template RNA into the exit channel. Therefore, EF-Tu in Qβ replicase modulates RNA elongation processes in a distinct manner from its established function in protein synthesis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of the initiation stage.
Figure 2: Structures of primary elongation stages.
Figure 3: Transition to the processive elongation stage.
Figure 4: Separation and translocation of RNAs and template RNA channels.
Figure 5: In vitro RNA polymerization by Qβ replicase variants.
Figure 6: Schematic representation of RNA polymerization by Qβ replicase.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Lai, M.M. Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244, 1–12 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Blumenthal, T. & Carmichael, G.G. RNA replication: function and structure of Qbeta replicase. Annu. Rev. Biochem. 48, 525–548 (1979).

    Article  CAS  PubMed  Google Scholar 

  3. Kondo, M., Gallerani, R. & Weissmann, C. Subunit structure of Qbeta replicase. Nature 228, 525–527 (1970).

    Article  CAS  PubMed  Google Scholar 

  4. Kamen, R. Characterization of the subunits of Qbeta replicase. Nature 228, 527–533 (1970).

    Article  CAS  PubMed  Google Scholar 

  5. Blumenthal, T., Landers, T.A. & Weber, K. Bacteriophage Q replicase contains the protein biosynthesis elongation factors EF Tu and EF Ts. Proc. Natl. Acad. Sci. USA 69, 1313–1317 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wahba, A.J. et al. Subunit I of G beta replicase and 30 S ribosomal protein S1 of Escherichia coli, Evidence for the identity of the two proteins. J. Biol. Chem. 249, 3314–3316 (1974).

    CAS  PubMed  Google Scholar 

  7. Kamen, R., Kondo, M., Römer, W. & Weissmann, C. Reconstitution of Q replicase lacking subunit with protein-synthesis-interference factor i. Eur. J. Biochem. 31, 44–51 (1972).

    Article  CAS  PubMed  Google Scholar 

  8. Hori, K., Harada, K. & Kuwano, M. Function of bacteriophage Qbeta replicase containing an altered subunit IV. J. Mol. Biol. 86, 699–708 (1974).

    Article  CAS  PubMed  Google Scholar 

  9. Landers, T.A., Blumenthal, T. & Weber, K. Function and structure in ribonucleic acid phage Q beta ribonucleic acid replicase. The roles of the different subunits in transcription of synthetic templates. J. Biol. Chem. 249, 5801–5808 (1974).

    CAS  PubMed  Google Scholar 

  10. Agirrezabala, X. & Frank, J. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu. Q. Rev. Biophys. 42, 159–200 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Schuette, J.C. et al. GTPase activation of elongation factor EF-Tu by the ribosome during decoding. EMBO J. 28, 755–765 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Schmeing, T.M. et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688–694 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Takeshita, D. & Tomita, K. Assembly of Qβ viral RNA polymerase with host translational elongation factors EF-Tu and -Ts. Proc. Natl. Acad. Sci. USA 107, 15733–15738 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Dahlberg, J.E. Terminal sequences of bacteriophage RNAs. Nature 220, 548–552 (1968).

    Article  CAS  PubMed  Google Scholar 

  15. Weissmann, C., Billeter, M.A., Goodman, H.M., Hindley, J. & Weber, H. Structure and function of phage RNA. Annu. Rev. Biochem. 42, 303–328 (1973).

    Article  CAS  PubMed  Google Scholar 

  16. Sprinzl, M. & Cramer, F. The -C-C-A end of tRNA and its role in protein biosynthesis. Prog. Nucleic Acid Res. Mol. Biol. 22, 1–69 (1979).

    Article  CAS  PubMed  Google Scholar 

  17. Silverman, P.M. Replication of RNA viruses: specific binding of the Q RNA polymerase to Q RNA. Arch. Biochem. Biophys. 157, 222–233 (1973).

    Article  CAS  PubMed  Google Scholar 

  18. Blumenthal, T. β replicase template specificity: different templates require different GTP concentrations for initiation. Proc. Natl. Acad. Sci. USA 77, 2601–2605 (1980).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kidmose, R.T., Vasiliev, N.N., Chetverin, A.B., Andersen, G.R. & Knudsen, C.R. Structure of the Qbeta replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins. Proc. Natl. Acad. Sci. USA 107, 10884–10889 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kawashima, T., Berthet-Colominas, C., Wulff, M., Cusack, S. & Leberman, R. The structure of the Escherichia coli EF-Tu:EF-Ts complex at 2.5 Å resolution. Nature 379, 511–518 (1996).

    Article  CAS  PubMed  Google Scholar 

  21. Caldas, T.D., Yaagoubi, A.E. & Richarme, G. Chaperone properties of bacterial elongation factor EF-Tu. J. Biol. Chem. 273, 11478–11482 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Kudlicki, W., Coffman, A., Kramer, G. & Hardesty, B. Renaturation of rhodanese by translational elongation factor (EF) Tu. J. Biol. Chem. 272, 32206–32210 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Suzuki, H., Ueda, T., Taguchi, H. & Takeuchi, N. Chaperone properties of mammalian mitochondrial translation elongation factor Tu. J. Biol. Chem. 282, 4076–4084 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Karring, H. et al. Qβ phage resistance by deletion of the coiled-coil motif in elongation factor Ts. J. Biol. Chem. 279, 1878–1884 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Brautigam, C.A. & Steitz, T.A. Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes. Curr. Opin. Struct. Biol. 8, 54–63 (1998).

    Article  CAS  PubMed  Google Scholar 

  26. Steitz, T.A. & Steitz, J.A. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498–6502 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Butcher, S.J., Grimes, J.M., Makeyev, E.V., Bamford, D.H. & Stuart, D.I. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235–240 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Salgado, P.S. et al. The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase. Structure 12, 307–316 (2004).

    CAS  PubMed  Google Scholar 

  29. Makeyev, E.V. & Bamford, D.H. Replicase activity of purified recombinant P2 of double-stranded RNA bacteriophage φ6. EMBO J. 19, 124–133 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Makeyev, E.V. & Bamford, D.H. The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J. 19, 6275–6284 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rensing, U. & August, J.T. The 3′-terminus and the replication of phage RNA. Nature 224, 853–856 (1969).

    Article  CAS  PubMed  Google Scholar 

  32. Schaffner, W., Ruegg, K.J. & Weissmann, C. Nanovariant RNAs: nucleotide sequence and interaction with bacteriophage Qβ replicase. J. Mol. Biol. 117, 877–907 (1977).

    Article  CAS  PubMed  Google Scholar 

  33. Nissen, P. et al. Crystal structure of the ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. Science 270, 1464–1472 (1995).

    Article  CAS  PubMed  Google Scholar 

  34. Nissen, P., Thirup, S., Kjeldgaard, M. & Nyborg, J. The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA. Structure 7, 143–156 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Nissen, P., Kjeldgaard, M., Thirup, S., Clark, B.F. & Nyborg, J. The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold. Biochimie 78, 921–933 (1996).

    Article  CAS  PubMed  Google Scholar 

  36. Gromadski, K.B., Wieden, H.J. & Rodnina, M.V. Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu. Biochemistry 41, 162–169 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Wang, Y., Jiang, Y., Meyering-Voss, M., Sprinzl, M. & Sigler, P.B. Crystal structure of the EF-Tu:EF-Ts complex from Thermus thermophilus. Nat. Struct. Biol. 4, 650–656 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Yin, Y.W. & Steitz, T.A. Structural basis for the transition from initiation to elongation transcription in T7 RNA polymerase. Science 298, 1387–1395 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Yin, Y.W. & Steitz, T.A. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 116, 393–404 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Hall, T.C. Transfer RNA-like structures in viral genomes. Int. Rev. Cytol. 60, 1–26 (1979).

    Article  CAS  PubMed  Google Scholar 

  41. Haenni, A.L., Joshi, S. & Chapeville, F. tRNA-like structures in the genomes of RNA viruses. Prog. Nucleic Acid Res. Mol. Biol. 27, 85–104 (1982).

    Article  CAS  PubMed  Google Scholar 

  42. Crick, F.H.C. The origin of the genetic code. J. Mol. Biol. 38, 367–379 (1968).

    Article  CAS  PubMed  Google Scholar 

  43. Orgel, L.E. Evolution of the genetic apparatus. J. Mol. Biol. 38, 381–393 (1968).

    Article  CAS  PubMed  Google Scholar 

  44. Weiner, A.M. & Maizels, N. tRNA-like structures tag the 3′ ends of genomic RNA molecules for replication: implications for the origin of protein synthesis. Proc. Natl. Acad. Sci. USA 84, 7383–7387 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zamora, H., Luce, R. & Biebricher, C.K. Design of artificial short-chained RNA species that are replicated by Q beta replicase. Biochemistry 34, 1261–1266 (1995).

    Article  CAS  PubMed  Google Scholar 

  46. Kita, H. et al. Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit. J. Biosci. Bioeng. 101, 421–426 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  48. Vagin, A. & Teplyakov, A. An approach to multi-copy search in molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 56, 1622–1624 (2000).

    Article  CAS  PubMed  Google Scholar 

  49. McRee, D.E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  51. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Hamada for technical assistance. We thank the beamline staff of BL-17A (KEK) for technical assistance during data collection. This work was supported by grants to K.T. from the Precursory Research for Embryonic Science and Technology program of the Japan Science and Technology Agency, the Funding program for Next Generation World-Leading Researchers (NEXT program) of the Japan Society for the Promotion of Science, the Takeda Science Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Cell Science Research Foundation.

Author information

Authors and Affiliations

  1. Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan

    Daijiro Takeshita & Kozo Tomita

  2. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Saitama, Japan

    Kozo Tomita

Authors
  1. Daijiro Takeshita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kozo Tomita
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.T. planned and designed the research, K.T. and D.T. designed the experiments, D.T. conducted the experiments and D.T. and K.T. analyzed the data, discussed the results and wrote the paper.

Corresponding author

Correspondence to Kozo Tomita.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Discussion (PDF 408 kb)

Supplementary Movie 1

Splitting the dsRNA of the template and growing RNAs. (MOV 1193 kb)

Supplementary Movie 2

The template entrance and exit channels in core Qβ replicase. (MOV 1122 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Takeshita, D., Tomita, K. Molecular basis for RNA polymerization by Qβ replicase. Nat Struct Mol Biol 19, 229–237 (2012). https://doi.org/10.1038/nsmb.2204

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2204

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing