Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel.
Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel.
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DOI：
10.1021/jacs.5b07910
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发表时间：
2015-12-23
影响因子：
15
通讯作者：
Gu LQ
中科院分区：
文献类型：
--
作者：
Zhang X;Xu X;Yang Z;Burcke AJ;Gates KS;Chen SJ;Gu LQ
假结是RNA的基本三级结构，在mRNA翻译调控中具有重要作用。光镊等分子力光谱方法可以通过从两端牵拉RNA链来跟踪假结的解折叠中间态，但这种方法诱导的动力学解折叠途径可能不同于体内的动力学解折叠途径，其发生在翻译过程中，从5′端到3′端进行。在这里，我们开发了一种核糖体模拟，纳米孔拉分析解剖的矢量解折叠机制的假结。纳米孔中的假结解折叠途径（从5′到3′端或以相反方向）可以由在5′或3′端连接到假结的DNA前导序列控制。DNA和RNA易位之间的不同纳米孔电导用作孔中解折叠RNA的位置和结构的标记。有了这个设计，我们提供的证据表明，假结展开是一个两步，多态，金属离子调节的过程取决于拉动方向。最值得注意的是，在两个方向上的解折叠受到第一螺旋结构域解压缩（第一步）的速率限制，第一螺旋结构域是在5′ → 3′方向上的Hacking-1和在3′ → 5′方向上的Hacking-2，这表明在任一拉动方向上的初始解折叠步骤需要克服由非典型三链体碱基对和同轴堆叠相互作用贡献的能量障碍以稳定三级结构。这些发现为RNA载体解折叠机制提供了新的见解，RNA载体解折叠机制在包括移码在内的生物学功能中发挥着重要作用。
Pseudoknots are a fundamental RNA tertiary structure with important roles in regulation of mRNA translation. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot’s unfolding intermediate states by pulling the RNA chain from both ends, but the kinetic unfolding pathway induced by this method may be different from that in vivo, which occurs during translation and proceeds from the 5′ to 3′ end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The pseudoknot unfolding pathway in the nanopore, either from the 5′ to 3′ end or in the reverse direction, can be controlled by a DNA leader that is attached to the pseudoknot at the 5′ or 3′ ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we provided evidence that the pseudoknot unfolding is a two-step, multistate, metal ion-regulated process depending on the pulling direction. Most notably, unfolding in both directions is rate-limited by the unzipping of the first helix domain (first step), which is Helix-1 in the 5′ → 3′ direction and Helix-2 in the 3′ → 5′ direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the noncanonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanisms, which play an important role in biological functions including frameshifting.