Fürtig Boris, Richter Christian, Schell Peter, Wenter Philipp, Pitsch Stefan, Schwalbe Harald
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Frankfurt, Germany.
RNA Biol. 2008 Jan-Mar;5(1):41-8. doi: 10.4161/rna.5.1.5704. Epub 2008 Feb 6.
In order to relate the conformational dynamics of the hammerhead ribozyme to its biological function the cleavage reaction catalyzed by the hammerhead ribozyme was monitored by time-resolved nuclear magnetic resonance (NMR) spectroscopy. For this purpose, the two nucleosides around the scissile phosphodiester bond were selectively (13)C labelled in multi-step organic syntheses starting from uniformly (13)C-labelled glucose. The phosphoamidites were incorporated using phosphoamidite chemistry in the hammerhead substrate strand. In addition, the 2'-OH group on the 5'-side of the hammerhead substrate strand was labelled with a photolabile protecting group. This labelling strategy enabled a detailed characterisation of the nucleotides around the scissile phosphodiester bond in the ground state conformation of the hammerhead ribozyme in the absence and presence of Mg(2+) ions as well as of the product state. Photochemical induction of the reaction in situ was further characterized by time-resolved NMR spectroscopy. The detailed structural and dynamic investigations revealed that the conformation of the hammerhead ribozyme is significantly affected by addition of Mg(2+) leading to an ensemble of conformations where dynamic transitions between energetically similar conformations occur on the ms-timescale in the presence of Mg(2+). The dynamic transitions are localized around the catalytic core. Cleavage from this ensemble cannot be described by mono-exponential kinetics but follows bi-exponential kinetics. A model is described to take into account these experimental data.
为了将锤头状核酶的构象动力学与其生物学功能联系起来，通过时间分辨核磁共振（NMR）光谱监测了锤头状核酶催化的切割反应。为此，从均匀（13）C标记的葡萄糖开始，在多步有机合成中对可切割磷酸二酯键周围的两个核苷进行了选择性（13）C标记。使用亚磷酰胺化学方法将亚磷酰胺掺入锤头状底物链中。此外，锤头状底物链5'侧的2'-OH基团用一个光不稳定保护基团进行了标记。这种标记策略能够在不存在和存在Mg(2+)离子以及产物状态下，对锤头状核酶基态构象中可切割磷酸二酯键周围的核苷酸进行详细表征。通过时间分辨NMR光谱进一步表征了原位反应的光化学诱导。详细的结构和动力学研究表明，添加Mg(2+)会显著影响锤头状核酶的构象，导致形成一组构象，在Mg(2+)存在下，能量相似的构象之间的动态转变在毫秒时间尺度上发生。动态转变集中在催化核心周围。从这一组构象中的切割不能用单指数动力学来描述，而是遵循双指数动力学。描述了一个模型来考虑这些实验数据。
