Skip to main content
SpringerLink
Log in

Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of β-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Ahlquist P, Luckow V, Kaesberg P: Complete nucleotide sequence of brome mosaic virus RNA 3. J Mol Biol 153: 23–38 (1981).

    PubMed  Google Scholar 

  2. Beier H, Barciszewska M, Krupp G, Mitnacht R, Gross HJ: UAG readthrough during TMV RNA translation: Isolation and sequence of two tRNAsTyr with suppressor activity from tobacco plants. EMBO J 3: 351–356 (1984).

    Google Scholar 

  3. Beier H, Barciszewska M, Sickinger HD: The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts. EMBO J 3: 1091–1096 (1984).

    Google Scholar 

  4. Boccara M, Hamilton WDO, Baulcombe DC: The organisation and interviral homologies of genes at the 3′ end of tobacco rattle virus RNA 1. EMBO J 5: 223–229 (1986).

    Google Scholar 

  5. Bouzoubaa S, Ziegler V, Beck D, Guilley H, Richards K, Jonard G: Nucleotide sequence of beet necrotic yellow vein virus RNA-2. J Gen Virol 67: 1689–1700 (1986).

    Google Scholar 

  6. Bradford M: A rapid and sensitive method for the quantification of microgram quantities of protein using the principles of protein-dye binding. Anal Biochem 72: 248–254 (1976).

    Article  PubMed  Google Scholar 

  7. Bradshaw HDJr, Parson WW, Sheffer M, Lioubin PJ, Mulvihill ER, Gordon MP: Design, construction, and use of an electroporator for plant protoplasts and animal cells. Anal Biochem 166: 342–348 (1987).

    PubMed  Google Scholar 

  8. Carrington JC, Morris TJ: Characterization of the cell-free translation products of carnation mottle virus genomic and subgenomic RNAs. Virology 144: 1–10 (1985).

    Google Scholar 

  9. Fang RX, Nagy F, Sivasubramaniam S, Chua NH: Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. Plant Cell 1: 141–150 (1989).

    Article  PubMed  Google Scholar 

  10. Gallie DR, Lucas WJ, Walbot V: Visualizing mRNA expression in plant protoplasts: Factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301–311 (1989).

    PubMed  Google Scholar 

  11. Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: The 5′-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucl Acids Res 15: 3257–3273 (1987).

    PubMed  Google Scholar 

  12. Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expression in vivo. Nucl Acids Res 15: 8693–8711 (1987).

    PubMed  Google Scholar 

  13. Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: Mutational analysis of the tobacco mosaic virus 5′-leader for altered ability to enhance translation. Nucl Acids Res 16: 883–893 (1988).

    PubMed  Google Scholar 

  14. Goelet P, Lomonosoff GP, Butler PJG, Akam ME, Gait MJ, Karn J: Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci USA 79: 5818–5822 (1982).

    PubMed  Google Scholar 

  15. Goldbach RW: Molecular evolution of plant RNA viruses. Ann Rev Phytopath 24: 289–310 (1986).

    Article  Google Scholar 

  16. Guilley H, Carrington JC, Balazs E, Jonard G, Richards K, Morris TJ: Nucleotide sequence and genome organizatio of carnation mottle virus RNA. Nucl Acids Res 13: 6663–6677 (1985).

    PubMed  Google Scholar 

  17. Guilley H, Dudley RK, Jonard G, Balazs E, Richards KE: Transcription of cauliflower mosaic virus DNA: Detection of promoter sequences, and characterization of transcripts. Cell 30: 763–773 (1982).

    PubMed  Google Scholar 

  18. Hamilton WDO, Boccara M, Robinson DJ, Baulcombe DC: The complete nucleotide sequence of tobacco rattle virus RNA-1. J Gen Virol 68: 2563–2575 (1987).

    PubMed  Google Scholar 

  19. Harrison BD, Robinson DJ: Tobraviruses. In: Van Regenmortel MHV, Fraenkel-Conrat H (eds) The Plant Viruses, Vol. 2, The rod-shaped plant viruses. Plenum Press, New York (1986)

    Google Scholar 

  20. Ishikawa M, Meshi T, Motoyoshi F, Takamatsu N, Okada Y: In vitro mutagenesis of the putative replicase genes of tobacco mosaic virus. Nucl Acids Res 14: 8291–8305 (1986).

    PubMed  Google Scholar 

  21. Iturriaga G, Jefferson RA, Bevan MW: Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco. Plant Cell 1: 381–390 (1989).

    PubMed  Google Scholar 

  22. Jacks T, Power MD, Masiarz FR, Luciw PA, Barr PJ, Varmus HE: Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331: 280–283 (1988).

    PubMed  Google Scholar 

  23. Jacks T, Varmus HE: Expression of the rous sarcoma virus pol gene by ribosomal frameshifting. Science 230: 1237–1242 (1985).

    PubMed  Google Scholar 

  24. Jefferson RA. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).

    Google Scholar 

  25. Jefferson RA, Burgess SM, Hirsch D: β-Glucuronidase from Escherichia coli as a gene-gusion marker. Proc Natl Acad Sci USA 83: 8447–8451 (1986).

    PubMed  Google Scholar 

  26. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    PubMed  Google Scholar 

  27. Jobling SA, Gehrke L: Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325: 622–625 (1987).

    Article  PubMed  Google Scholar 

  28. Jones DS, Nemoto F, Kuchino Y, Masuda M, Yoshikura H, Nishimura S: The effect of specific mutations at and around the gag-pol junction of Moloney murine leukaemia virus. Nucl Acids Res 17: 5933–5945 (1989).

    PubMed  Google Scholar 

  29. Kamer G, Argos P: Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucl Acids Res 12: 7269–7282 (1984).

    PubMed  Google Scholar 

  30. Kavanagh TA, Jefferson RA, Bevan MW: Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein. Mol Gen Genet 215: 38–45 (1988).

    PubMed  Google Scholar 

  31. Kay R, Chan A, Daly M, McPherson J: Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302 (1987).

    Google Scholar 

  32. Koiwai A, Noguchi M, Tamaki E: Changes in the amino acid composition of tobacco cells in suspension culture. Phytochem 10: 561–566 (1971).

    Google Scholar 

  33. Koper-Zwarthoff EC, Brederode FT, Veeneman G, van Boom JH, Bol JF: Nucleotide sequences at the 5′ termini of the alfalfa mosaic virus RNAs and the intercistronic junction in RNA 3. Nucl Acids Res 8: 5635–5647 (1980).

    PubMed  Google Scholar 

  34. Kozak M: The scanning model for translation: An update. J Cell Biol 108: 229–241 (1989).

    PubMed  Google Scholar 

  35. Mandeles S: Location of unique sequences in tobacco mosaic virus ribonucleic acid. J Biol Chem 243: 3671–3674 (1968).

    PubMed  Google Scholar 

  36. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982).

    Google Scholar 

  37. Miller WA, Waterhouse PM, Gerlach WL: Sequence and organization of barley yellow dwarf genomic RNA. Nucl Acids Res 16: 6097–6111 (1988).

    PubMed  Google Scholar 

  38. Morch MD, Boyer JC, Haenni AL: Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA. Nucl Acids Res 16: 6157–6173 (1988).

    PubMed  Google Scholar 

  39. Morch MD, Drugeon G, Benicourt C: Analysis of the in vitro coding properties of the 3′ region of turnip yellow mosaic virus genomic RNA. Virology 119: 193–198 (1982).

    Article  Google Scholar 

  40. Murgola EJ, Hijazi KA, Goringer HU, Dahlberg AE: Mutant 16S ribosomal RNA: A codon-specific translational suppressor. Proc Natl Acad Sci USA 85: 4162–4165 (1988).

    PubMed  Google Scholar 

  41. Nutter RC, Scheets K, Panganiban LC, Lommel SA: The complete nucleotide sequence of the maize chlorotic mottle virus genome. Nucl Acids Res 17: 3163–3177 (1989).

    PubMed  Google Scholar 

  42. O'Connor M, Gesteland RF, Atkins JF: tRNA hopping: Enhancement by an expanded anticodon. EMBO J 8: 4315–4323 (1989).

    PubMed  Google Scholar 

  43. Odell JT, Knowlton S, Lin W, Mauvais CJ: Properties of an isolated transcription stimulating sequence derived from the cauliflower mosaic virus 35S promoter. Plant Mol Biol 10: 263–273 (1987).

    Google Scholar 

  44. Ohno T, Aoyagi M, Yamanashi Y, Saito H, Ikawa S, Meshi T, Okada Y: Nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome. J Biochem 96: 1915–1923 (1984).

    PubMed  Google Scholar 

  45. Ow DW, Jacobs JD, Howell SH: Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity. Proc Natl Acad Sci USA 84: 4870–4874 (1987).

    Google Scholar 

  46. Paterson R, Knight CA: Protein synthesis in tobacco protoplasts infected with tobacco mosaic virus. Virology 64: 10–22 (1975).

    Article  PubMed  Google Scholar 

  47. Pelham HRB: Leaky UAG termination codon in tobacco mosaic virus RNA. Nature 272: 469–471 (1978).

    PubMed  Google Scholar 

  48. Pelham HRB: Translation of tobacco rattle virus RNAs in vitro: Four proteins from three RNAs. Virology 97: 256–265 (1979).

    Article  Google Scholar 

  49. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    PubMed  Google Scholar 

  50. Siegel A, Hari V, Kolacz K: The effect of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts. Virology 85: 494–503 (1978).

    Article  PubMed  Google Scholar 

  51. Shen Z, Fox TD: Substitution of an invariant nucleotide at the base of the highly conserved ‘530-loop’ of 15S rRNA causes suppression of yeast mitochondrial ochre mutations. Nucl Acids Res 17: 4535–4539 (1989).

    PubMed  Google Scholar 

  52. Veidt I, Lot H, Leiser H, Scheidecker D, Guilley H, Richards K, Jonard G: Nucleotide sequence of beet western yellows virus RNA. Nucl Acids Res 16: 9917–9932 (1988).

    PubMed  Google Scholar 

  53. Weiss RB, Dunn DM, Atkins JF, Gesteland RF: Slippery runs, shifty stops, backward steps, and forward hops: −2, −1, +1, +5, and +6 ribosomal frameshifting. CSH Symp Quant Biol 52: 687–693 (1987).

    Google Scholar 

  54. Xiong Z, Lommel SA: The complete nucleotide sequence and genome organization of red vlover necrotic mosaic virus RNA-1. Virology 171: 543–554 (1989).

    Article  PubMed  Google Scholar 

  55. Ziegler V, Richards K, Guilley H, Jonard G, Putz C: Cell-free translation of beet necrotic yellow vein virus: Readthrough of the coat protein cistron. J Gen Virol 66: 2079–2087 (1985).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Howard Hughes Medical Institute, The University of Utah School of Medicine, 743 Wintrobe Bldg., 84132, Salt Lake City, UT, USA

    James M. Skuzeski, Lindy M. Nichols & Raymond F. Gesteland

  2. Department of Human Genetics, The University of Utah School of Medicine, 743 Wintrobe Bldg., 84132, Salt Lake City, UT, USA

    James M. Skuzeski, Lindy M. Nichols & Raymond F. Gesteland

Authors
  1. James M. Skuzeski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lindy M. Nichols
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Raymond F. Gesteland
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Skuzeski, J.M., Nichols, L.M. & Gesteland, R.F. Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors. Plant Mol Biol 15, 65–79 (1990). https://doi.org/10.1007/BF00017725

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00017725

Key words

Search

Navigation