Department of Biochemistry and Molecular Biology; Michigan State University; East Lansing, MI USA.
RNA Biol. 2013 Mar;10(3):425-35. doi: 10.4161/rna.23609. Epub 2013 Jan 16.
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(NH 3) 6 (3+). 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.
发夹核酶是一种原型的小分子、自我切割的 RNA 基序。它以包含两个相邻臂上内部环的四向 RNA 连接的形式自然存在。在化学催化之前，这两个环通过阳离子驱动的对接步骤相互作用，形成一个紧密整合的结构，每个环的构象在对接时发生剧烈变化。我们使用位于不同分子上的环 A 和环 B 的构建体来研究对接过程的热力学和动力学。使用一种新颖的 CD 差异测定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由亚毫摩尔浓度的交换惰性 Co(NH 3) 6 (3+)驱动。RNA 自我切割需要与具有比对接过程本身更高的表观协同性的低亲和力离子结合，这意味着，即使金属离子不直接与 RNA 配位，它们在发夹核酶功能中也发挥着催化作用，不仅仅是驱动环-环对接。表面等离子体共振测定法揭示了分子缔合的速度非常缓慢，考虑到相对紧密的环-环相互作用。这一观察结果与“双构象捕获”模型一致，其中只有同时处于小的、对接相容构象的环 A 和环 B 分子之间的碰撞才能产生结合的产物。
