Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Dec;11(13):5129–5135. doi: 10.1002/j.1460-2075.1992.tb05620.x

In vitro recombination and terminal elongation of RNA by Q beta replicase.

C K Biebricher 1, R Luce 1
PMCID: PMC556991  PMID: 1281452

Abstract

SV-11 is a short-chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV-11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV-11 contains an inverse duplication of the high-melting domain of MNV-11. SV-11 is thus a recombinant between the plus and minus strands of MNV-11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin-form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3' terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3' terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template-instructed synthesis. The reaction also adds nucleotides to the 3' terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.

Full text

PDF
5129

Images in this article

5130Image
on p.5130
5131Image
on p.5131
5132Image
on p.5132

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allison R., Thompson C., Ahlquist P. Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1820–1824. doi: 10.1073/pnas.87.5.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biebricher C. K., Diekmann S., Luce R. Structural analysis of self-replicating RNA synthesized by Qbeta replicase. J Mol Biol. 1982 Feb 5;154(4):629–648. doi: 10.1016/s0022-2836(82)80019-3. [DOI] [PubMed] [Google Scholar]
  3. Biebricher C. K., Eigen M., Gardiner W. C., Jr Kinetics of RNA replication: competition and selection among self-replicating RNA species. Biochemistry. 1985 Nov 5;24(23):6550–6560. doi: 10.1021/bi00344a037. [DOI] [PubMed] [Google Scholar]
  4. Biebricher C. K., Eigen M., Luce R. Product analysis of RNA generated de novo by Q beta replicase. J Mol Biol. 1981 Jun 5;148(4):369–390. doi: 10.1016/0022-2836(81)90182-0. [DOI] [PubMed] [Google Scholar]
  5. Biebricher C. K., Eigen M., Luce R. Template-free RNA synthesis by Q beta replicase. Nature. 1986 May 1;321(6065):89–91. doi: 10.1038/321089a0. [DOI] [PubMed] [Google Scholar]
  6. Biebricher C. K. Replication and evolution of short-chained RNA species replicated by Q beta replicase. Cold Spring Harb Symp Quant Biol. 1987;52:299–306. doi: 10.1101/sqb.1987.052.01.035. [DOI] [PubMed] [Google Scholar]
  7. Blumenthal T. Q beta replicase template specificity: different templates require different GTP concentrations for initiation. Proc Natl Acad Sci U S A. 1980 May;77(5):2601–2605. doi: 10.1073/pnas.77.5.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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 U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feix G., Hake H. Primer directed initiation of RNA synthesis catalysed by Qbeta replicase. Biochem Biophys Res Commun. 1975 Jul 22;65(2):503–509. doi: 10.1016/s0006-291x(75)80176-8. [DOI] [PubMed] [Google Scholar]
  11. Feix G. Primer-dependent copying of rabbit globin mRNA with Qbeta replicase. Nature. 1976 Feb 19;259(5544):593–594. doi: 10.1038/259593a0. [DOI] [PubMed] [Google Scholar]
  12. Feix G., Sano H. Initiation specificity of the poly(cytidylic acid)-dependent Qbeta replicase activity. Eur J Biochem. 1975 Oct 1;58(1):59–64. doi: 10.1111/j.1432-1033.1975.tb02348.x. [DOI] [PubMed] [Google Scholar]
  13. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  15. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  16. King A. M., McCahon D., Slade W. R., Newman J. W. Recombination in RNA. Cell. 1982 Jul;29(3):921–928. doi: 10.1016/0092-8674(82)90454-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kramer F. R., Mills D. R., Cole P. E., Nishihara T., Spiegelman S. Evolution in vitro: sequence and phenotype of a mutant RNA resistant to ethidium bromide. J Mol Biol. 1974 Nov 15;89(4):719–736. doi: 10.1016/0022-2836(74)90047-3. [DOI] [PubMed] [Google Scholar]
  18. Lai M. M., Baric R. S., Makino S., Keck J. G., Egbert J., Leibowitz J. L., Stohlman S. A. Recombination between nonsegmented RNA genomes of murine coronaviruses. J Virol. 1985 Nov;56(2):449–456. doi: 10.1128/jvi.56.2.449-456.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lai M. M. RNA recombination in animal and plant viruses. Microbiol Rev. 1992 Mar;56(1):61–79. doi: 10.1128/mr.56.1.61-79.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Levisohn R., Spiegelman S. Further extracellular Darwinian experiments with replicating RNA molecules: diverse variants isolated under different selective conditions. Proc Natl Acad Sci U S A. 1969 Jul;63(3):805–811. doi: 10.1073/pnas.63.3.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levisohn R., Spiegelman S. The cloning of a self-replicating RNA molecule. Proc Natl Acad Sci U S A. 1968 Jul;60(3):866–872. doi: 10.1073/pnas.60.3.866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loss P., Schmitz M., Steger G., Riesner D. Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J. 1991 Mar;10(3):719–727. doi: 10.1002/j.1460-2075.1991.tb08002.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mills D. R., Kramer F. R. Structure-independent nucleotide sequence analysis. Proc Natl Acad Sci U S A. 1979 May;76(5):2232–2235. doi: 10.1073/pnas.76.5.2232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mills D. R., Peterson R. L., Spiegelman S. An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc Natl Acad Sci U S A. 1967 Jul;58(1):217–224. doi: 10.1073/pnas.58.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Munishkin A. V., Voronin L. A., Chetverin A. B. An in vivo recombinant RNA capable of autocatalytic synthesis by Q beta replicase. Nature. 1988 Jun 2;333(6172):473–475. doi: 10.1038/333473a0. [DOI] [PubMed] [Google Scholar]
  27. Obinata M., Nasser D. S., McCarthy B. J. Synthesis of probes for RNA using Qbeta-replicase. Biochem Biophys Res Commun. 1975 May 19;64(2):640–647. doi: 10.1016/0006-291x(75)90369-1. [DOI] [PubMed] [Google Scholar]
  28. Palasingam K., Shaklee P. N. Reversion of Q beta RNA phage mutants by homologous RNA recombination. J Virol. 1992 Apr;66(4):2435–2442. doi: 10.1128/jvi.66.4.2435-2442.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Palmenberg A., Kaesberg P. Synthesis of complementary strands of heterologous RNAs with Qbeta replicase. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1371–1375. doi: 10.1073/pnas.71.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Saffhill R., Schneider-Bernloehr H., Orgel L. E., Spiegelman S. In vitro selection of bacteriophage Q-beta ribonucleic acid variants resistant to ethidium bromide. J Mol Biol. 1970 Aug;51(3):531–539. doi: 10.1016/0022-2836(70)90006-9. [DOI] [PubMed] [Google Scholar]
  31. Sanger F., Brownlee G. G., Barrell B. G. A two-dimensional fractionation procedure for radioactive nucleotides. J Mol Biol. 1965 Sep;13(2):373–398. doi: 10.1016/s0022-2836(65)80104-8. [DOI] [PubMed] [Google Scholar]
  32. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sippel A. E. Purification and characterization of adenosine triphosphate: ribonucleic acid adenyltransferase from Escherichia coli. Eur J Biochem. 1973 Aug 1;37(1):31–40. doi: 10.1111/j.1432-1033.1973.tb02953.x. [DOI] [PubMed] [Google Scholar]
  34. Sumper M., Luce R. Evidence for de novo production of self-replicating and environmentally adapted RNA structures by bacteriophage Qbeta replicase. Proc Natl Acad Sci U S A. 1975 Jan;72(1):162–166. doi: 10.1073/pnas.72.1.162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Van Tol H., Buzayan J. M., Bruening G. Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus. Virology. 1991 Jan;180(1):23–30. doi: 10.1016/0042-6822(91)90005-v. [DOI] [PubMed] [Google Scholar]
  37. Vournakis J. N., Carmichael G. G., Efstratiadis A. Synthesis of RNA complementary of rabbit globin mRNA by Qbeta replicase. Biochem Biophys Res Commun. 1976 Jun 7;70(3):774–782. doi: 10.1016/0006-291x(76)90659-8. [DOI] [PubMed] [Google Scholar]
  38. Weber H., Weissmann C. The 3'-termini of bacteriophage Q-beta plus and minus strands. J Mol Biol. 1970 Jul 28;51(2):215–224. doi: 10.1016/0022-2836(70)90138-5. [DOI] [PubMed] [Google Scholar]
  39. Weiss B. G., Schlesinger S. Recombination between Sindbis virus RNAs. J Virol. 1991 Aug;65(8):4017–4025. doi: 10.1128/jvi.65.8.4017-4025.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Woodson S. A., Cech T. R. Reverse self-splicing of the tetrahymena group I intron: implication for the directionality of splicing and for intron transposition. Cell. 1989 Apr 21;57(2):335–345. doi: 10.1016/0092-8674(89)90971-9. [DOI] [PubMed] [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  42. Zaug A. J., Cech T. R. The intervening sequence RNA of Tetrahymena is an enzyme. Science. 1986 Jan 31;231(4737):470–475. doi: 10.1126/science.3941911. [DOI] [PubMed] [Google Scholar]
  43. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. van der Kuyl A. C., Neeleman L., Bol J. F. Complementation and recombination between alfalfa mosaic virus RNA3 mutants in tobacco plants. Virology. 1991 Aug;183(2):731–738. doi: 10.1016/0042-6822(91)91002-X. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES