Kinetic analysis of the RNAi enzyme complex
Kinetic analysis of the RNAi enzyme complex
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DOI：
10.1038/nsmb780
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发表时间：
2004-07-01
影响因子：
16.8
通讯作者：
Zamore, PD
中科院分区：
文献类型：
--
作者：
Haley, B;Zamore, PD
SiRNA指导的核糖核蛋白复合体RISC在RNA干扰途径中催化目标RNA的切割。在这里，我们证明了在ATP存在下，siRNA编程的RISC是一个经典的Michaelis-Menten酶。在没有ATP的情况下，多轮催化的速度受到酶裂解产物的释放的限制。动力学分析表明，siRNA的不同区域在靶标识别、切割和产物释放的循环中发挥着不同的作用。靠近siRNA 5末端的碱基对靶RNA结合能的贡献不成比例，而由siRNA的中心和3个区域形成的碱基对提供了催化所需的螺旋几何形状。最后，在siRNA与其RNA靶相遇之前，剪刀状磷酸在靶RNA上的位置似乎是在RISC组装过程中确定的。
The siRNA-directed ribonucleoprotein complex, RISC, catalyzes target RNA cleavage in the RNA interference pathway. Here, we show that siRNA-programmed RISC is a classical Michaelis-Menten enzyme in the presence of ATP. In the absence of ATP, the rate of multiple rounds of catalysis is limited by release of the cleaved products from the enzyme. Kinetic analysis suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release. Bases near the siRNA 5 end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3 regions of the siRNA provide a helical geometry required for catalysis. Finally, the position of the scissile phosphate on the target RNA seems to be determined during RISC assembly, before the siRNA encounters its RNA target.