Walter F, Murchie A I, Thomson J B, Lilley D M
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University, Dundee, UK.
Biochemistry. 1998 Oct 6;37(40):14195-203. doi: 10.1021/bi981513+.
The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.
发夹状核酶的天然形式由一个四臂RNA连接体组成，其中携带单链环的螺旋是相邻的臂。该连接体可被视为构建活性核酶的框架，并且切割速率可通过改变连接体的构象来调节。我们发现基于连接体形式的发夹状核酶在镁离子、钙离子或锶离子中具有活性，但在锰离子、镉离子或钠离子中无活性。利用荧光共振能量转移实验，我们研究了核酶的整体结构。构建体的基本折叠基于成对的螺旋堆积，因此两个携带环的臂位于相对的堆积螺旋对上。在镁离子、钙离子或锶离子存在的情况下，核酶的连接体发生旋转，形成扭曲的反平行结构，使两个环之间产生紧密的物理接触。锰离子诱导相同的整体折叠，但无催化活性；因此，这种整体构象的变化对于催化活性是必要的，但不是充分的。将构象对离子浓度的依赖性拟合到二态模型表明，需要两个离子的协同结合来实现折叠。然而，切割活性还需要进一步的离子结合。六氨合钴离子也能引起整体折叠，而亚精胺则产生一种更对称的反平行结构形式。镉离子产生一种不同的折叠形式，可解释为在连接体未折叠时环与环之间的紧密缔合。钠离子不能诱导核酶发生任何折叠，核酶仍保持轻微平行状态。这些结果与由两个IIA族金属离子结合诱导的折叠过程一致，这两个离子分布在连接体和环界面之间。
