Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance
Single-molecule enzymology of RNA: Essential functional groups impact catalysis from a distance
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DOI：
10.1073/pnas.0403575101
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发表时间：
2004-07-06
影响因子：
11.1
通讯作者：
Walter, NG
中科院分区：
文献类型：
--
作者：
Rueda, D;Bokinsky, G;Walter, NG
发夹核酶是研究RNA折叠和功能的一个极简范例。在这种酶中，两个结构域通过诱导配合对接，形成一个催化核心，介导特定的骨干裂解反应。在这里，我们完全剖析了其可逆反应途径，包括两个结构转变（对接/解对接）和一个化学步骤（裂解/连接），通过应用单分子荧光共振能量转移（FRET）分析、系综裂解分析和动力学模拟的组合。这使我们能够量化远离催化位点的基本官能团的修饰对单个速率常数的影响。我们发现所有的核酶变体都显示出相似的分割成有效的非交换分子亚群，具有不同的分离速率常数。这导致了异质切割活性，通常观察到的RNA酶。在区域连接处的修改还会导致异构对接。令人惊讶的是，大多数修饰不仅影响对接/解对接，而且显著影响距离催化位点相当远的内部化学速率常数。我们提出，一个耦合的分子运动网络将RNA的遥远部分与其反应位点连接起来，这表明RNA和蛋白质酶之间存在先前描述的类比。我们的研究结果也对诸如活性位点远端的药物和配体的作用或变构成RNA的工程等应用具有广泛的意义。
The hairpin ribozyme is a minimalist paradigm for studying RNA folding and function. In this enzyme, two domains dock by induced fit to form a catalytic core that mediates a specific backbone cleavage reaction. Here, we have fully dissected its reversible reaction pathway, which comprises two structural transitions (docking/undocking) and a chemistry step (cleavage/ligation), by applying a combination of single-molecule fluorescence resonance energy transfer (FRET) assays, ensemble cleavage assays, and kinetic simulations. This has allowed us to quantify the effects that modifications of essential functional groups remote from the site of catalysis have on the individual rate constants. We find that all ribozyme variants show similar fractionations into effectively non-interchanging molecule subpopulations of distinct undocking rate constants. This leads to heterogeneous cleavage activity as commonly observed for RNA enzymes. A modification at the domain junction additionally leads to heterogeneous docking. Surprisingly, most modifications not only affect docking/undocking but also significantly impact the internal chemistry rate constants over a substantial distance from the site of catalysis. We propose that a network of coupled molecular motions connects distant parts of the RNA with its reaction site, which suggests a previously undescribed analogy between RNA and protein enzymes. Our findings also have broad implications for applications such as the action of drugs and ligands distal to the active site or the engineering of allostery into RNA.