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Real ribozymes suggest a relaxed error threshold


The error threshold for replication, the critical copying fidelity below which the fittest genotype deterministically disappears, limits the length of the genome that can be maintained by selection. Primordial replication must have been error-prone, and so early replicators are thought to have been necessarily short1. The error threshold also depends on the fitness landscape. In an RNA world2, many neutral and compensatory mutations can raise the threshold, below which the functional phenotype3, rather than a particular sequence, is still present4,5. Here we show, on the basis of comparative analysis of two extensively mutagenized ribozymes, that with a copying fidelity of 0.999 per digit per replication the phenotypic error threshold rises well above 7,000 nucleotides, which permits the selective maintenance of a functionally rich riboorganism6 with a genome of more than 100 different genes, the size of a tRNA. This requires an order of magnitude of improvement in the accuracy of in vitro–generated polymerase ribozymes7,8. Incidentally, this genome size coincides with that estimated for a minimal cell achieved by top-down analysis9, omitting the genes dealing with translation.

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Figure 1: Mutagenized ribozymes.
Figure 2: Time to extinction in generations as a function of the per digit effective mutation rate (μ*) in a population of constant size with N = 10,000 molecules for the Neurospora VS ribozyme (a,c,e,g) and the hairpin ribozyme (b,d,f,h).
Figure 3: Fraction of mutants with full activity (filled circles) or any activity (open squares), as a function of the number of point mutations.
Figure 4: Relationship between the per digit replication accuracy (q) and the permissible genome size (L) estimated from equation 2 with λ = 0.22 and s = 351.


  1. Eigen, M. Self organization of matter and the evolution of biological macromolecules. Naturwissenschaften 10, 465–523 (1971).

    Article  Google Scholar 

  2. Gilbert, W. The RNA world. Nature 319, 618 (1986).

    Article  Google Scholar 

  3. Huynen, M.A., Stadler, P.F. & Fontana, W. Smoothness within ruggedness: the role of neutrality in adaptation. Proc. Natl. Acad. Sci. USA 93, 397–401 (1996).

    Article  CAS  Google Scholar 

  4. Reidys, C., Forst, C.V. & Schuster, P. Replication and mutation on neutral networks. Bull. Math. Biol. 63, 57–94 (2001).

    Article  CAS  Google Scholar 

  5. Takeuchi, N., Poorthuis, P.H. & Hogeweg, P. Phenotypic error threshold; additivity and epistasis in RNA evolution. BMC Evol. Biol. 5, 9 (2005).

    Article  Google Scholar 

  6. Jeffares, D.C., Poole, A.M. & Penny, D. Relics from the RNA world. J. Mol. Evol. 46, 18–36 (1998).

    Article  CAS  Google Scholar 

  7. Johnston, W.K., Unrau, P.J., Lawrence, M.S., Glasen, M.E. & Bartel, D.P. RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science 292, 1319–1325 (2001).

    Article  CAS  Google Scholar 

  8. Müller, U.F. & Bartel, D.P. Substrate 2′-hydroxyl groups required for ribozyme-catalyzed polymerization. Chem. Biol. 10, 799–806 (2003).

    Article  Google Scholar 

  9. Gil, R., Silva, F.J., Peretó, J. & Moya, A. Determination of the core of a minimal bacterial gene set. Microbiol. Mol. Biol. Rev. 68, 518–537 (2004).

    Article  CAS  Google Scholar 

  10. Maynard Smith, J. Hypercycles and the origin of life. Nature 20, 445–446 (1979).

    Article  Google Scholar 

  11. Maynard Smith, J. Models of evolution. Proc. R. Soc. Lond. B 219, 315–325 (1983).

    Article  Google Scholar 

  12. Maynard Smith, J. & Szathmáry, E. The Major Transitions in Evolution (Oxford Univ. Press, Oxford, 1995).

    Google Scholar 

  13. Benner, B., Ellington, A.D., Ge, L., Gasfeld, A. & Leanz, G.F. Natural selection, protein enzgineering, and the last riboorganism: rational model building in biochemistry. Cold Spring Harb. Symp. Quant. Biol. 52, 56–63 (1987).

    Article  Google Scholar 

  14. Fedor, M. Structure and function of the hairpin ribozyme. J. Mol. Biol. 297, 269–291 (2000).

    Article  CAS  Google Scholar 

  15. Lafontaine, D.A., Norman, D.G. & Lilley, D.M.J. The structure and active site of the Varkund satellite ribozyme. Biochem. Soc. Trans. 30, 1170–1175 (2002).

    Article  CAS  Google Scholar 

  16. Lehman, N. & Joyce, G.F. Evolution in vitro: analysis of a lineage of ribozymes. Curr. Biol. 3, 723–734 (1993).

    Article  CAS  Google Scholar 

  17. Takeda, Y., Sarai, A. & Rivera, V.M. Analysis of the sequence-specific interactions between Cro repressor and operator DNA by systematic base substitution experiments. Proc. Natl. Acad. Sci. USA 86, 439–443 (1989).

    Article  CAS  Google Scholar 

  18. Sandberg, W.S. & Terwilliger, T.C. Engineering multiple properties of a protein by combinatorial mutagenesis. Proc. Natl. Acad. Sci. USA 90, 8367–8371 (1993).

    Article  CAS  Google Scholar 

  19. Skinner, M.M. & Terwilliger, T.C. Potential use of additivity of mutational effects in simplifying protein engineering. Proc. Natl. Acad. Sci. USA 93, 10753–10757 (1996).

    Article  CAS  Google Scholar 

  20. Eigen, M., McCaskill, J.S. & Schuster, P. The molecular quasispecies. Adv. Chem. Phys. 75, 149–263 (1989).

    CAS  Google Scholar 

  21. Santos, M., Zintzaras, E. & Szathmáry, E. Recombination in primeval genomes: a step forward but still a long leap from maintaining a sizeable genome. J. Mol. Evol. 59, 507–519 (2004).

    Article  CAS  Google Scholar 

  22. Domingo, E. & Holland, J.J. in The Evolutionary Biology of Viruses (ed. Morse, S.S.) 161–183 (Raven, New York, 1994).

    Google Scholar 

  23. Hofacker, I.L. et al. Fast folding and comparison of RNA secondary structures. Monatsh. Chem. 125, 167–188 (1994).

    Article  CAS  Google Scholar 

  24. Hofacker, I.L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003).

    Article  CAS  Google Scholar 

  25. Beattie, T.L., Olive, J.E. & Collins, R.A. A secondary-structure model for the self-cleaving region of the Neurospora VS RNA. Proc. Natl. Acad. Sci. USA 92, 4686–4690 (1995).

    Article  CAS  Google Scholar 

  26. Butcher, S.E. & Burke, J.M. Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex. J. Mol. Biol. 244, 52–63 (1994).

    Article  CAS  Google Scholar 

  27. Butcher, S.E. & Burke, J.M. A photo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme. Biochemistry 33, 992–999 (1994).

    Article  CAS  Google Scholar 

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We thank I. Hofacker for his help with the Vienna RNA package, P. Mezey for allowing us to use the computer facilities at MUN (Canada) and F. Kondrashov for comments on an earlier version of the manuscript. Computer facilities were provided by Microdigit. This work was supported by grant and postdoctoral fellowship from the Hungarian National Research Fund (OTKA) to Á.K. M.S. is partially supported by Fundación Ramón Areces (Spain). This work was also supported by the COST D27 action (Prebiotic chemistry and early evolution).

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Correspondence to Eörs Szathmáry.

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Supplementary information

Supplementary Table 1

Mutations of the Neurospora VS ribozyme. (PDF 35 kb)

Supplementary Table 2

Mutations of the Hairpin ribozyme. (PDF 32 kb)

Supplementary Note

Construction of the empirically-supported fitness landscape for the Neurospora VS and the hairpin ribozyme. (PDF 58 kb)

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Kun, Á., Santos, M. & Szathmáry, E. Real ribozymes suggest a relaxed error threshold. Nat Genet 37, 1008–1011 (2005).

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