Significant change in the structure of a ribozyme upon introduction of a phosphorothioate linkage at P9: NMR reveals a conformational fluctuation in the core region of a hammerhead ribozyme

Ken Ichi Suzumura, Masaki Warashina, Koichi Yoshinari, Yoshiyuki Tanaka, Tomoko Kuwabara, Masaya Orita, Kazunari Taira

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

A modified hammerhead ribozyme (R32S) with a phosphorothioate linkage between G8 and A9, a site that is considered to play a crucial role in catalysis, was examined by high-resolution 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Signals due to imino protons that corresponded to stems were observed, but the anticipated signals due to imino protons adjacent to the phosphorothioate linkage were not detected and the 31P signal due to the phosphorothioate linkage was also absent irrespective of the presence or absence of the substrate. 31P NMR is known to reflect backbone mobility, and thus the absence of signals indicated that the introduction of sulfur at P9 had increased the mobility of the backbone near the phosphorothioate linkage. The addition of metal ions did not regenerate the signals that had disappeared, a result that implied that the structure of the core region of the hammerhead ribozyme had fluctuated even in the presence of metal ions. Furthermore, kinetic analysis suggested that most of the R32S-substrate complexes generated in the absence of Mg2+ ions were still in an inactive form and that Mg2+ ions induced a further conformational change that converted such complexes to an activated state. Finally, according to available NMR studies, signals due to the imino protons of the central core region that includes the P9 metal binding site were broadened or not observed, suggesting that this catalytically important region might be intrinsically flexible. Our present analysis revealed a significant change in the structure of the ribozyme upon the introduction of the single phosphorothioate linkage at P9 that is in general considered to be a conservative modification. Copyright (C) 2000 Federation of European Biochemical Societies.

Original languageEnglish
Pages (from-to)106-112
Number of pages7
JournalFEBS Letters
Volume473
Issue number1
DOIs
Publication statusPublished - 2000 May 4

Keywords

  • Conformational change
  • Hammerhead ribozyme
  • Kinetics
  • Nuclear magnetic resonance
  • Phosphorothioate

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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