Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.
Intermolecular domain docking in the hairpin ribozyme: metal dependence, binding kinetics and catalysis.
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DOI：
10.4161/rna.23609
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发表时间：
2013-03
期刊：
影响因子：
4.1
通讯作者：
Hoogstraten CG
中科院分区：
文献类型：
--
作者：
Sumita M;White NA;Julien KR;Hoogstraten CG
发夹状核酶是一个小的、自我切割的RNA基序的原型。它自然地以四向RNA连接的形式存在，在相邻的手臂上包含两个内部环。这两个环在化学催化之前以阳离子驱动的对接步骤相互作用，形成一个紧密结合的结构，对接后每个环的构象都发生了戏剧性的变化。我们使用环A和环B驻留在单独的分子上的结构来研究对接过程的热力学和动力学。使用一种新的CD差分法来分离与结构域对接相关的金属离子的影响，我们发现分子间对接过程是由交换惰性Co(NH3)63+的亚毫摩尔浓度驱动的。RNA的自切割需要与低亲和力的离子结合，比对接过程本身具有更大的表观协同性，这意味着，即使在没有与RNA直接配位的情况下，金属离子在发夹状核酶功能中也起到了催化作用，而不仅仅是驱动环-环对接。表面等离子体共振分析显示，考虑到相对紧密的环-环相互作用，分子结合非常缓慢。这一观察结果与“双构象捕获”模型是一致的，在该模型中，只有同时处于次要的、可对接的构象中的环A和环B分子之间的碰撞才能产生结合。
The hairpin ribozyme is a prototype small, self-cleaving RNA motif. It exists naturally as a four-way RNA junction containing two internal loops on adjoining arms. These two loops interact in a cation-driven docking step prior to chemical catalysis to form a tightly integrated structure, with dramatic changes occurring in the conformation of each loop upon docking. We investigate the thermodynamics and kinetics of the docking process using constructs in which loop A and loop B reside on separate molecules. Using a novel CD difference assay to isolate the effects of metal ions linked to domain docking, we find the intermolecular docking process to be driven by sub-millimolar concentrations of the exchange-inert Co(NH3)63+. RNA self-cleavage requires binding of lower-affinity ions with greater apparent cooperativity than the docking process itself, implying that, even in the absence of direct coordination to RNA, metal ions play a catalytic role in hairpin ribozyme function beyond simply driving loop-loop docking. Surface plasmon resonance assays reveal remarkably slow molecular association, given the relatively tight loop-loop interaction. This observation is consistent with a “double conformational capture” model in which only collisions between loop A and loop B molecules that are simultaneously in minor, docking-competent conformations are productive for binding.