Walter F, Murchie A I, Lilley D M
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, U.K.
Biochemistry. 1998 Dec 15;37(50):17629-36. doi: 10.1021/bi9821115.
The hairpin ribozyme consists of two loop-carrying duplexes (called A and B) that are adjacent arms of a four-way junction in its natural context in the viral RNA. We have shown previously that the activity of the ribozyme is strongly influenced by the structure adopted by the junction. In this study, we have used fluorescence resonance energy transfer to analyze the conformation and folding of the isolated four-way junction. Like other four-way RNA junctions, in the absence of added metal ions this junction adopts a square configuration of coaxially stacked arms, based on A on D and B on C stacking. Upon addition of magnesium ions, the junction undergoes an ion-induced transition to an antiparallel conformation. The data are consistent with folding induced by the binding of a single ion, with an apparent association constant in the range of 2000 M-1. Other divalent metal ions (calcium or manganese) can also induce this change in structure; however, sodium ions are unable to substitute for these ions, and are slightly inhibitory with respect to the transition. The loop-free hairpin junction adopts the same stacking conformer as the full ribozyme, but forms a more symmetrical X-shaped structure. In addition, the apparent stoichiometry of structural ion binding is lower for the isolated junction, and the affinity is considerably lower.
发夹状核酶由两个带有环的双链体（称为A和B）组成，在病毒RNA的自然环境中，它们是一个四向连接点的相邻臂。我们之前已经表明，核酶的活性受到连接点所采用结构的强烈影响。在本研究中，我们利用荧光共振能量转移来分析分离出的四向连接点的构象和折叠情况。与其他四向RNA连接点一样，在不添加金属离子的情况下，这个连接点基于A与D以及B与C的堆叠，采用同轴堆叠臂的方形构型。加入镁离子后，连接点会发生离子诱导的转变，形成反平行构象。数据与单个离子结合诱导的折叠情况一致，其表观缔合常数在2000 M⁻¹范围内。其他二价金属离子（钙或锰）也能诱导这种结构变化；然而，钠离子不能替代这些离子，并且对这种转变有轻微抑制作用。无环发夹状连接点采用与完整核酶相同的堆叠构象异构体，但形成更对称的X形结构。此外，对于分离出的连接点，结构离子结合的表观化学计量较低，且亲和力也低得多。
