Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Feb 25;20(4):747–753. doi: 10.1093/nar/20.4.747

Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions.

Y A Suh 1, P K Kumar 1, F Nishikawa 1, E Kayano 1, S Nakai 1, O Odai 1, S Uesugi 1, K Taira 1, S Nishikawa 1
PMCID: PMC312013  PMID: 1542571

Abstract

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Full text

PDF
747

Images in this article

751Image
on p.751

Selected References

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

  1. Belinsky M. G., Dinter-Gottlieb G. Non-ribozyme sequences enhance self-cleavage of ribozymes derived from Hepatitis delta virus. Nucleic Acids Res. 1991 Feb 11;19(3):559–564. doi: 10.1093/nar/19.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branch A. D., Robertson H. D. Efficient trans cleavage and a common structural motif for the ribozymes of the human hepatitis delta agent. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10163–10167. doi: 10.1073/pnas.88.22.10163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Endo Y., Glück A., Chan Y. L., Tsurugi K., Wool I. G. RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function. J Biol Chem. 1990 Feb 5;265(4):2216–2222. [PubMed] [Google Scholar]
  4. Endo Y., Tsurugi K. RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem. 1987 Jun 15;262(17):8128–8130. [PubMed] [Google Scholar]
  5. Govindarajan S., Chin K. P., Redeker A. G., Peters R. L. Fulminant B viral hepatitis: role of delta agent. Gastroenterology. 1984 Jun;86(6):1417–1420. [PubMed] [Google Scholar]
  6. Hampel A., Tritz R., Hicks M., Cruz P. 'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res. 1990 Jan 25;18(2):299–304. doi: 10.1093/nar/18.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kuo M. Y., Sharmeen L., Dinter-Gottlieb G., Taylor J. Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol. 1988 Dec;62(12):4439–4444. doi: 10.1128/jvi.62.12.4439-4444.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Makino S., Chang M. F., Shieh C. K., Kamahora T., Vannier D. M., Govindarajan S., Lai M. M. Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA. Nature. 1987 Sep 24;329(6137):343–346. doi: 10.1038/329343a0. [DOI] [PubMed] [Google Scholar]
  9. Nakamaye K. L., Eckstein F. Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1986 Dec 22;14(24):9679–9698. doi: 10.1093/nar/14.24.9679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Perreault J. P., Labuda D., Usman N., Yang J. H., Cedergren R. Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis. Biochemistry. 1991 Apr 23;30(16):4020–4025. doi: 10.1021/bi00230a029. [DOI] [PubMed] [Google Scholar]
  11. Perreault J. P., Wu T. F., Cousineau B., Ogilvie K. K., Cedergren R. Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature. 1990 Apr 5;344(6266):565–567. doi: 10.1038/344565a0. [DOI] [PubMed] [Google Scholar]
  12. Perrotta A. T., Been M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature. 1991 Apr 4;350(6317):434–436. doi: 10.1038/350434a0. [DOI] [PubMed] [Google Scholar]
  13. Perrotta A. T., Been M. D. The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant. Nucleic Acids Res. 1990 Dec 11;18(23):6821–6827. doi: 10.1093/nar/18.23.6821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  15. Rosenstein S. P., Been M. D. Evidence that genomic and antigenomic RNA self-cleaving elements from hepatitis delta virus have similar secondary structures. Nucleic Acids Res. 1991 Oct 11;19(19):5409–5416. doi: 10.1093/nar/19.19.5409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosenstein S. P., Been M. D. Self-cleavage of hepatitis delta virus genomic strand RNA is enhanced under partially denaturing conditions. Biochemistry. 1990 Sep 4;29(35):8011–8016. doi: 10.1021/bi00487a002. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Smith J. B., Dinter-Gottlieb G. Antigenomic Hepatitis delta virus ribozymes self-cleave in 18 M formamide. Nucleic Acids Res. 1991 Mar 25;19(6):1285–1289. doi: 10.1093/nar/19.6.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Symons R. H. Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends Biochem Sci. 1989 Nov;14(11):445–450. doi: 10.1016/0968-0004(89)90103-5. [DOI] [PubMed] [Google Scholar]
  20. Taira K., Uchimaru T., Tanabe K., Uebayasi M., Nishikawa S. Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate. Nucleic Acids Res. 1991 May 25;19(10):2747–2753. doi: 10.1093/nar/19.10.2747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Taira K., Uebayasi M., Maeda H., Furukawa K. Energetics of RNA cleavage: implications for the mechanism of action of ribozymes. Protein Eng. 1990 Aug;3(8):691–701. doi: 10.1093/protein/3.8.691. [DOI] [PubMed] [Google Scholar]
  22. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taylor J. W., Schmidt W., Cosstick R., Okruszek A., Eckstein F. The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8749–8764. doi: 10.1093/nar/13.24.8749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thill G., Blumenfeld M., Lescure F., Vasseur M. Self-cleavage of a 71 nucleotide-long ribozyme derived from hepatitis delta virus genomic RNA. Nucleic Acids Res. 1991 Dec 11;19(23):6519–6525. doi: 10.1093/nar/19.23.6519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Uebayasi M., Uchimaru T., Tanabe K., Nishikawa S., Taira K. Preferential chelation of cationic ligands to axial-equatorial oxygens over equatorial-equatorial dianionic oxygens: implication to the mechanism of action of ribozymes. Nucleic Acids Symp Ser. 1991;(25):107–108. [PubMed] [Google Scholar]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. Wu H. N., Lai M. M. RNA conformational requirements of self-cleavage of hepatitis delta virus RNA. Mol Cell Biol. 1990 Oct;10(10):5575–5579. doi: 10.1128/mcb.10.10.5575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wu H. N., Lin Y. J., Lin F. P., Makino S., Chang M. F., Lai M. M. Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1831–1835. doi: 10.1073/pnas.86.6.1831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yang J. H., Perreault J. P., Labuda D., Usman N., Cedergren R. Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme. Biochemistry. 1990 Dec 25;29(51):11156–11160. doi: 10.1021/bi00503a002. [DOI] [PubMed] [Google Scholar]

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

RESOURCES