KINETICS OF INTERMOLECULAR CLEAVAGE BY HAMMERHEAD RIBOZYMES
KINETICS OF INTERMOLECULAR CLEAVAGE BY HAMMERHEAD RIBOZYMES
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DOI：
10.1021/bi00163a012
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发表时间：
1992-12-08
期刊：
影响因子：
2.9
通讯作者：
UHLENBECK, OC
中科院分区：
文献类型：
--
作者：
FEDOR, MJ;UHLENBECK, OC
锤头状催化RNA通过酯交换机制切割RNA的磷酸二酯骨架，产生具有2 '-3'-环状磷酸和5 '-羟基末端的产物。分子间锤头切割反应的最小动力学机制包括底物结合、切割和产物释放。这些步骤的元素速率常数测定与六个锤头序列。底物长度和序列的变化对裂解步骤的速率几乎没有影响，但在需要螺旋-卷曲转变的底物解离和产物释放步骤中观察到显著差异。底物结合和产物解离的速率与基于简单RNA双链体行为的预测很好地相关，但底物解离速率明显快于预期。消除催化活性的核酶和底物改变增加了锤头复合物的稳定性。这些结果表明，底物不稳定可能在锤头状催化中发挥作用。
The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.