Fast cleavage kinetics of a natural hammerhead ribozyme
Fast cleavage kinetics of a natural hammerhead ribozyme
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DOI：
10.1021/ja046848v
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发表时间：
2004-09-08
影响因子：
15
通讯作者：
Pardi, A
中科院分区：
文献类型：
--
作者：
Canny, MD;Jucker, FM;Pardi, A
锤头状核酶是一种催化RNA切割和连接的小RNA基序。充分研究的最小锤头状基序在生理条件下是无活性的，并且需要高Mg 2+浓度以进行有效切割。相比之下，天然锤头鲨在生理条件下是活性的，并且在催化核心之外含有降低对Mg 2+的需求的基序。本文采用单转换动力学方法研究了曼氏血吸虫锤头状核酶的反式切割结构对Mg ~（2+）和pH的依赖性。与最小的锤头状基序相比，天然的Schistosomaribozyme需要少100倍的Mg 2+才能实现1 min-1的切割速率。改进的催化作用是由于茎I和II中的环之间的三级相互作用，并且可能是由于活性构象的数量增加而引起的。在最佳pH和Mg 2+条件下，该核酶在25 °C下以超过870 min-1的速度切割，进一步证明了该核酶令人印象深刻的催化能力。
The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg2+concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg2+. Single-turnover kinetics were used here to characterize the Mg2+and pH dependence for cleavage of a trans-cleaving construct of theSchistosoma mansoninatural hammerhead ribozyme. Compared to the minimal hammerhead motif, the naturalSchistosomaribozyme requires 100-fold less Mg2+to achieve a cleavage rate of 1 min-1. The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg2+conditions this ribozyme cleaves at over 870 min-1at 25 °C, further demonstrating the impressive catalytic power of this ribozyme.