Wilson Timothy J, Nahas Michelle, Araki Lisa, Harusawa Shinya, Ha Taekjip, Lilley David M J
Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee DD1 5EH, UK.
Blood Cells Mol Dis. 2007 Jan-Feb;38(1):8-14. doi: 10.1016/j.bcmd.2006.10.004. Epub 2006 Dec 4.
The nucleolytic ribozymes catalyse site-specific phosphodiester cleavage and ligation transesterification reactions in RNA. The hairpin ribozyme folds to generate an intimate loop-loop interaction to create the local environment in which catalysis can proceed. We have studied the ion-induced folding using single-molecule FRET experiments, showing that the four-way helical junction accelerates the folding 500-fold by introducing a discrete intermediate that juxtaposes the loops. Using FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions, free of uncertainties in the contributions of docking and substrate dissociation processes. On average, the cleaved ribozyme undergoes several docking-undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G8 and A38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with pKa=6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid-base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both.
核酶催化RNA中的位点特异性磷酸二酯键切割和连接转酯反应。发夹状核酶折叠形成紧密的环-环相互作用，以创造能够进行催化反应的局部环境。我们通过单分子荧光共振能量转移（FRET）实验研究了离子诱导的折叠过程，结果表明，四链螺旋连接通过引入一个使环并列的离散中间体，使折叠速度加快了500倍。利用FRET，我们可以观察单个发夹状核酶分子经历多次切割和连接循环的过程，并测量内部反应的速率，而不受对接和底物解离过程贡献的不确定性影响。平均而言，切割后的核酶在发生连接反应之前会经历几次对接-解对接事件。基于这些实验，我们探讨了核碱基G8和A38在催化过程中的作用。切割和连接反应均依赖于pH值，这与pKa = 6.2的基团的滴定相对应。我们使用了一种新型核糖核苷，其中这些碱基被咪唑取代，以研究酸碱催化在这种核酶中的作用。我们观察到了显著的切割和连接速率，并且两者的pH依赖性均呈钟形。
