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. 1998 Feb 15;26(4):980–987. doi: 10.1093/nar/26.4.980

The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine.

S R Lynch 1, I Tinoco Jr 1
PMCID: PMC147372  PMID: 9461457

Abstract

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.

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Selected References

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  1. Batey R. T., Battiste J. L., Williamson J. R. Preparation of isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol. 1995;261:300–322. doi: 10.1016/s0076-6879(95)61015-4. [DOI] [PubMed] [Google Scholar]
  2. Been M. D., Perrotta A. T., Rosenstein S. P. Secondary structure of the self-cleaving RNA of hepatitis delta virus: applications to catalytic RNA design. Biochemistry. 1992 Dec 1;31(47):11843–11852. doi: 10.1021/bi00162a024. [DOI] [PubMed] [Google Scholar]
  3. Cai Z., Tinoco I., Jr Solution structure of loop A from the hairpin ribozyme from tobacco ringspot virus satellite. Biochemistry. 1996 May 14;35(19):6026–6036. doi: 10.1021/bi952985g. [DOI] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Dieckmann T., Feigon J. Assignment methodology for larger RNA oligonucleotides: application to an ATP-binding RNA aptamer. J Biomol NMR. 1997 Apr;9(3):259–272. doi: 10.1023/a:1018622708674. [DOI] [PubMed] [Google Scholar]
  6. Haschemeyer A. E., Rich A. Nucleoside conformations: an analysis of steric barriers to rotation about the glycosidic bond. J Mol Biol. 1967 Jul 28;27(2):369–384. doi: 10.1016/0022-2836(67)90026-5. [DOI] [PubMed] [Google Scholar]
  7. Haynie S. L., Whitesides G. M. Preparation of a mixture of nucleoside triphosphates suitable for use in synthesis of nucleotide phosphate sugars from ribonucleic acid using nuclease P1, a mixture of nucleoside monophosphokinases and acetate kinase. Appl Biochem Biotechnol. 1990 Mar;23(3):205–220. doi: 10.1007/BF02942055. [DOI] [PubMed] [Google Scholar]
  8. Holbrook S. R., Cheong C., Tinoco I., Jr, Kim S. H. Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs. Nature. 1991 Oct 10;353(6344):579–581. doi: 10.1038/353579a0. [DOI] [PubMed] [Google Scholar]
  9. Ippel J. H., Lanzotti V., Galeone A., Mayol L., van den Boogaart J. E., Pikkemaat J. A., Altona C. Conformation of the circular dumbbell d<pCGC-TT-GCG-TT>: structure determination and molecular dynamics. J Biomol NMR. 1995 Dec;6(4):403–422. doi: 10.1007/BF00197639. [DOI] [PubMed] [Google Scholar]
  10. Kolk M. H., Heus H. A., Hilbers C. W. The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution. EMBO J. 1997 Jun 16;16(12):3685–3692. doi: 10.1093/emboj/16.12.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Nikonowicz E. P., Sirr A., Legault P., Jucker F. M., Baer L. M., Pardi A. Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 1992 Sep 11;20(17):4507–4513. doi: 10.1093/nar/20.17.4507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Perrotta A. T., Been M. D. Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme. Nucleic Acids Res. 1993 Aug 25;21(17):3959–3965. doi: 10.1093/nar/21.17.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Perrotta A. T., Been M. D. Core sequences and a cleavage site wobble pair required for HDV antigenomic ribozyme self-cleavage. Nucleic Acids Res. 1996 Apr 1;24(7):1314–1321. doi: 10.1093/nar/24.7.1314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Puttaraju M., Perrotta A. T., Been M. D. A circular trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res. 1993 Sep 11;21(18):4253–4258. doi: 10.1093/nar/21.18.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Schroth G. P., Kagawa T. F., Ho P. S. Structure and thermodynamics of nonalternating C.G base pairs in Z-DNA: the 1.3-A crystal structure of the asymmetric hexanucleotide d(m5CGGGm5CG).d(m5CGCCm5CG). Biochemistry. 1993 Dec 14;32(49):13381–13392. doi: 10.1021/bi00212a002. [DOI] [PubMed] [Google Scholar]
  19. Sklenár V., Miyashiro H., Zon G., Miles H. T., Bax A. Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy. FEBS Lett. 1986 Nov 10;208(1):94–98. doi: 10.1016/0014-5793(86)81539-3. [DOI] [PubMed] [Google Scholar]
  20. Sklenár V., Peterson R. D., Rejante M. R., Feigon J. Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments. J Biomol NMR. 1994 Jan;4(1):117–122. doi: 10.1007/BF00178339. [DOI] [PubMed] [Google Scholar]
  21. Thill G., Vasseur M., Tanner N. K. Structural and sequence elements required for the self-cleaving activity of the hepatitis delta virus ribozyme. Biochemistry. 1993 Apr 27;32(16):4254–4262. doi: 10.1021/bi00067a013. [DOI] [PubMed] [Google Scholar]
  22. Wang A. H., Gessner R. V., van der Marel G. A., van Boom J. H., Rich A. Crystal structure of Z-DNA without an alternating purine-pyrimidine sequence. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3611–3615. doi: 10.1073/pnas.82.11.3611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wijmenga S. S., Heus H. A., Werten B., van der Marel G. A., van Boom J. H., Hilbers C. W. Assignment strategies and analysis of cross-peak patterns and intensities in the three-dimensional homonuclear TOCSY-NOESY of RNA. J Magn Reson B. 1994 Feb;103(2):134–141. doi: 10.1006/jmrb.1994.1021. [DOI] [PubMed] [Google Scholar]
  24. 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]

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