Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Dec 25;20(24):6681–6686. doi: 10.1093/nar/20.24.6681

Generation of viroid conformational isomers that are stable to incubation with magnesium ions and in a nuclear extract from tomato plants.

U Pace 1, A D Branch 1, H D Robertson 1
PMCID: PMC334586  PMID: 1282703

Abstract

We identified conditions for heating and quick cooling viroid RNAs in the presence of salt which lead to the production of conformational isomers stable to incubation for at least 45 minutes at 30 degrees in the presence of magnesium ions. Elution in 0.3 M NaCl allowed the purification of an electrophoretically slow form of an in vitro transcript carrying a complete copy of the potato spindle tuber viroid RNA sequence. Slow forms of this transcript and of kinase-labeled linear viroid RNA persisted for longer than 20 minutes when incubated with a protein-rich extract prepared from the nuclei of uninfected tomato plants, although both were slowly cleaved by a nuclease present in this extract. The fast form of the transcript was highly resistant to this tomato ribonuclease. The slow form of the transcript was much more susceptible to cleavage by RNase T1 than the fast form of this RNA, suggesting that the reduced gel mobility of the slow forms results from their relatively open structure. The ability to purify viroid conformational isomers from polyacrylamide gels will facilitate biochemical studies aimed at identifying host components interacting with RNAs of the viroid replication complex, which may not all be present in the most thermodynamically favored rodlike structure of mature viroids.

Full text

PDF
6681

Images in this article

6683Image
on p.6683
6683Image
on p.6683
6683Image
on p.6683
6684Image
on p.6684
6684Image
on p.6684
6685Image
on p.6685
6685Image
on p.6685

Selected References

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

  1. Andreazzoli M., Gerbi S. A. Changes in 7SL RNA conformation during the signal recognition particle cycle. EMBO J. 1991 Apr;10(4):767–777. doi: 10.1002/j.1460-2075.1991.tb08008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branch A. D., Benenfeld B. J., Paul C. P., Robertson H. D. Analysis of ultraviolet-induced RNA-RNA cross-links: a means for probing RNA structure-function relationships. Methods Enzymol. 1989;180:418–442. doi: 10.1016/0076-6879(89)80115-6. [DOI] [PubMed] [Google Scholar]
  3. Branch A. D., Benenfeld B. J., Robertson H. D. Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6590–6594. doi: 10.1073/pnas.82.19.6590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Branch A. D., Robertson H. D. A replication cycle for viroids and other small infectious RNA's. Science. 1984 Feb 3;223(4635):450–455. doi: 10.1126/science.6197756. [DOI] [PubMed] [Google Scholar]
  5. Diener T. O. Viroid processing: a model involving the central conserved region and hairpin I. Proc Natl Acad Sci U S A. 1986 Jan;83(1):58–62. doi: 10.1073/pnas.83.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  7. Franklin R. M. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1504–1511. doi: 10.1073/pnas.55.6.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gross H. J., Domdey H., Lossow C., Jank P., Raba M., Alberty H., Sänger H. L. Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature. 1978 May 18;273(5659):203–208. doi: 10.1038/273203a0. [DOI] [PubMed] [Google Scholar]
  9. Hecker R., Wang Z. M., Steger G., Riesner D. Analysis of RNA structures by temperature-gradient gel electrophoresis: viroid replication and processing. Gene. 1988 Dec 10;72(1-2):59–74. doi: 10.1016/0378-1119(88)90128-x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Noller H. F. Ribosomal RNA and translation. Annu Rev Biochem. 1991;60:191–227. doi: 10.1146/annurev.bi.60.070191.001203. [DOI] [PubMed] [Google Scholar]
  12. Riesner D., Henco K., Rokohl U., Klotz G., Kleinschmidt A. K., Domdey H., Jank P., Gross H. J., Sänger H. L. Structure and structure formation of viroids. J Mol Biol. 1979 Sep 5;133(1):85–115. doi: 10.1016/0022-2836(79)90252-3. [DOI] [PubMed] [Google Scholar]
  13. Riesner D. Viroids: from thermodynamics to cellular structure and function. Mol Plant Microbe Interact. 1991 Mar-Apr;4(2):122–131. doi: 10.1094/mpmi-4-122. [DOI] [PubMed] [Google Scholar]
  14. Rizzetto M., Bonino F., Verme G. Hepatitis delta virus infection of the liver: progress in virology, pathobiology, and diagnosis. Semin Liver Dis. 1988 Nov;8(4):350–356. doi: 10.1055/s-2008-1040556. [DOI] [PubMed] [Google Scholar]
  15. Schumacher J., Sänger H. L., Riesner D. Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue. EMBO J. 1983;2(9):1549–1555. doi: 10.1002/j.1460-2075.1983.tb01622.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sharmeen L., Kuo M. Y., Dinter-Gottlieb G., Taylor J. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J Virol. 1988 Aug;62(8):2674–2679. doi: 10.1128/jvi.62.8.2674-2679.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sogo J. M., Koller T., Diener T. O. Potato spindle tuber viroid. X. Visualization and size determination by electron microscopy. Virology. 1973 Sep;55(1):70–80. doi: 10.1016/s0042-6822(73)81009-8. [DOI] [PubMed] [Google Scholar]
  18. Tabler M., Sänger H. L. Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious. EMBO J. 1984 Dec 20;3(13):3055–3062. doi: 10.1002/j.1460-2075.1984.tb02257.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tsagris M., Tabler M., Mühlbach H. P., Sänger H. L. Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells. EMBO J. 1987 Aug;6(8):2173–2183. doi: 10.1002/j.1460-2075.1987.tb02488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Visvader J. E., Forster A. C., Symons R. H. Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors. Nucleic Acids Res. 1985 Aug 26;13(16):5843–5856. doi: 10.1093/nar/13.16.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. van Wezenbeek P., Vos P., van Boom J., van Kammen A. Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA. Nucleic Acids Res. 1982 Dec 20;10(24):7947–7957. doi: 10.1093/nar/10.24.7947. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES