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

FEBS Letters

Taira, Kazunari¹  Suzumura, Ken-ichi²  Yoshinari, Koichi¹  Kuwabara, Tomoko¹  Orita, Masaya²  Tanaka, Yoshiyuki¹  Warashina, Masaki¹ 

[1] National Institute for Advanced Interdisciplinary Research, Tsukuba Science City 305-8562, Japan;Yamanouchi Pharmaceutical Co. Ltd., 21 Miyukigaoka, Tsukuba Science City 305-8585, Japan

关键词: Hammerhead ribozyme;    Nuclear magnetic resonance;    Conformational change;    Phosphorothioate;    Kinetics;   

DOI  :  10.1016/S0014-5793(00)01499-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown