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.

  • Letter
  • Published:

An equilibrium-dependent retroviral mRNA switch regulates translational recoding

Nature volume 480, pages 561–564 (2011)Cite this article

Subjects

Abstract

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly1,2. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon3. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio4,5,6,7,8. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.

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: MLV-PK conformational-equilibrium-dependent read-through.
Figure 2: Functional data for modulation of the equilibrium.
Figure 3: Compensatory effects of the S1–L2 turn on the engineered inter-helical bend and equilibrium-based frameshifting levels.
Figure 4: Model for equilibrium-based mechanism.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and restraints for the final ensemble of ten structures of PKinactive have been deposited in the Protein Data Bank under accession code 2LC8.

References

  1. Felsenstein, K. M. & Goff, S. P. Expression of the Gag-Pol fusion protein of Moloney murine leukemia virus without Gag protein does not induce virion formation or proteolytic processing. J. Virol. 62, 2179–2182 (1988)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Shehu-Xhilaga, M., Crowe, S. M. & Mak, J. Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity. J. Virol. 75, 1834–1841 (2001)

    Article  CAS  Google Scholar 

  3. Baranov, P. V., Gesteland, R. F. & Atkins, J. F. Recoding: translational bifurcations in gene expression. Gene 286, 187–201 (2002)

    Article  CAS  Google Scholar 

  4. Philipson, L. et al. Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: enhancement of the Gag-Pol polypeptide with yeast suppressor tRNA. Cell 13, 189–199 (1978)

    Article  CAS  Google Scholar 

  5. Yoshinaka, Y., Katoh, I., Copeland, T. D. & Oroszlan, S. Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon. Proc. Natl Acad. Sci. USA 82, 1618–1622 (1985)

    Article  ADS  CAS  Google Scholar 

  6. Wills, N. M., Gesteland, R. F. & Atkins, J. F. Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon. Proc. Natl Acad. Sci. USA 88, 6991–6995 (1991)

    Article  ADS  CAS  Google Scholar 

  7. Wills, N. M., Gesteland, R. F. & Atkins, J. F. Pseudoknot-dependent read-through of retroviral gag termination codons: importance of sequences in the spacer and loop 2. EMBO J. 13, 4137–4144 (1994)

    Article  CAS  Google Scholar 

  8. Alam, S. L., Wills, N. M., Ingram, J. A., Atkins, J. F. & Gesteland, R. F. Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus. J. Mol. Biol. 288, 837–852 (1999)

    Article  CAS  Google Scholar 

  9. Brierley, I., Pennell, S. & Gilbert, R. J. Viral RNA pseudoknots: versatile motifs in gene expression and replication. Nature Rev. Microbiol. 5, 598–610 (2007)

    Article  CAS  Google Scholar 

  10. Honigman, A., Wolf, D., Yaish, S., Falk, H. & Panet, A. cis Acting RNA sequences control the gag-pol translation readthrough in murine leukemia virus. Virology 183, 313–319 (1991)

    Article  CAS  Google Scholar 

  11. Feng, Y. X., Yuan, H., Rein, A. & Levin, J. G. Bipartite signal for read-through suppression in murine leukemia virus mRNA: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an RNA pseudoknot. J. Virol. 66, 5127–5132 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Nixon, P. L. & Giedroc, D. P. Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot. J. Mol. Biol. 296, 659–671 (2000)

    Article  CAS  Google Scholar 

  13. Grentzmann, G., Ingram, J. A., Kelly, P. J., Gesteland, R. F. & Atkins, J. F. A dual-luciferase reporter system for studying recoding signals. RNA 4, 479–486 (1998)

    Article  CAS  Google Scholar 

  14. Schroeder, S., Kim, J. & Turner, D. H. G. A. and U.U mismatches can stabilize RNA internal loops of three nucleotides. Biochemistry 35, 16105–16109 (1996)

    Article  CAS  Google Scholar 

  15. Badhwar, J., Karri, S., Cass, C. K., Wunderlich, E. L. & Znosko, B. M. Thermodynamic characterization of RNA duplexes containing naturally occurring 1 × 2 nucleotide internal loops. Biochemistry 46, 14715–14724 (2007)

    Article  CAS  Google Scholar 

  16. Michiels, P. J. et al. Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol. 310, 1109–1123 (2001)

    Article  CAS  Google Scholar 

  17. Clanton-Arrowood, K., McGurk, J. & Schroeder, S. J. 3′ terminal nucleotides determine thermodynamic stabilities of mismatches at the ends of RNA helices. Biochemistry 47, 13418–13427 (2008)

    Article  CAS  Google Scholar 

  18. Cornish, P. V., Hennig, M. & Giedroc, D. P. A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated −1 ribosomal frameshifting. Proc. Natl Acad. Sci. USA 102, 12694–12699 (2005)

    Article  ADS  CAS  Google Scholar 

  19. Atkins, J. F. & Gesteland, R. F. Recoding: Expansion of Decoding Rules Enriches Gene Expression (Springer, 2010)

    Book  Google Scholar 

  20. Al-Hashimi, H. M. & Walter, N. G. RNA dynamics: it is about time. Curr. Opin. Struct. Biol. 18, 321–329 (2008)

    Article  CAS  Google Scholar 

  21. Namy, O., Moran, S. J., Stuart, D. I., Gilbert, R. J. & Brierley, I. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature 441, 244–247 (2006)

    Article  ADS  CAS  Google Scholar 

  22. Giedroc, D. P. & Cornish, P. V. Frameshifting RNA pseudoknots: structure and mechanism. Virus Res. 139, 193–208 (2009)

    Article  CAS  Google Scholar 

  23. 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  Google Scholar 

  24. D’Souza, V., Dey, A., Habib, D. & Summers, M. F. NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus. J. Mol. Biol. 337, 427–442 (2004)

    Article  Google Scholar 

  25. Güntert, P., Mumenthaler, C. & Wüthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997)

    Article  Google Scholar 

  26. Kuhlman, B., Luisi, D. L., Young, P. & Raleigh, D. P. pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions. Biochemistry 38, 4896–4903 (1999)

    Article  CAS  Google Scholar 

  27. Legault, P. & Pardi, A. In-situ probing of adenine protonation in RNA by 13C NMR. J. Am. Chem. Soc. 116, 8390–8391 (1994)

    Article  CAS  Google Scholar 

  28. Staple, D. W. & Butcher, S. E. Solution structure and thermodynamic investigation of the HIV-1 frameshift inducing element. J. Mol. Biol. 349, 1011–1023 (2005)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Atkins for the p2luc plasmid, the New York Structural Biology Center for NMR time and A. Hawkins, S. Leiman and J. Gullinger for their contributions. B.H.-L. and S.P.G. would also like to thank the late D. Wolf for his inspiration and many helpful discussions. S.P.G. acknowledges grant R37 CA30488 from the NCI/NIH and is an Investigator with the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, 10032, New York, USA

    Brian Houck-Loomis & Stephen P. Goff

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, 02138,, Massachusetts, USA

    Michael A. Durney, Carolina Salguero, Neelaabh Shankar, Julia M. Nagle & Victoria M. D’Souza

Authors
  1. Brian Houck-Loomis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael A. Durney
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carolina Salguero
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Neelaabh Shankar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Julia M. Nagle
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephen P. Goff
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Victoria M. D’Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.M.D’S., B.H.-L., M.A.D. and S.P.G. conceived of and designed the experiments. V.M.D’S., M.A.D., C.S., N.S. and B.H.-L. did the structural analysis, B.H.-L. did the recoding assays, and M.A.D. and J.M.N. did the in vivo assay for the double mutant. M.A.D. and V.M.D’S. wrote the manuscript with assistance from B.H.-L.

Corresponding author

Correspondence to Victoria M. D’Souza.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Text, Supplementary References, Supplementary Table 1 and Supplementary Figures 1-14 with legends. (PDF 7353 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Houck-Loomis, B., Durney, M., Salguero, C. et al. An equilibrium-dependent retroviral mRNA switch regulates translational recoding. Nature 480, 561–564 (2011). https://doi.org/10.1038/nature10657

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10657

This article is cited by

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.

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