Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme
Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme
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DOI：
10.1073/pnas.95.11.6091
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发表时间：
1998-05-26
影响因子：
11.1
通讯作者：
Burke, JM
中科院分区：
文献类型：
--
作者：
Esteban, JA;Walter, NG;Burke, JM
发夹状核酶催化的RNA裂解反应为两相动力学，Chase实验表明，反应的慢相是由于可逆底物与非活性构象异构体结合所致。为了研究多相动力学的结构基础，我们开发了一种酶促RNA修饰方法，该方法选择性地捕获结合到非活性构象上的底物，并允许使用物理和动力学策略来分离和分析两种形式的核酶-底物复合体。通过在切割反应中加入T4RNA连接酶，捕获了该复合体的非活性形式，导致底物的5‘端与核酶的3’端的共价键，并选择性和定量地消融了反应的缓慢动力学相。这一结果表明，非活性形式的核酶-底物复合体可以采用螺旋2和3同轴堆积的构象，而活性形式不能获得这种构象，因为螺旋连接处的急剧弯曲可能是由催化活性所需的结构域间三级接触稳定的。这些结果被用来通过在两个结构域之间设计新的界面来提高发夹状核酶的活性，一个包含非核苷酸邻苯键，另一个用三向连接取代双向连接。这些修饰的核酶中的每一个都优先采用活性构象，并表现出更高的催化效率。
The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5' end of the substrate to the 3' end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.