Nuclear Magnetic Resonance Structure of the III-IV-V Three-Way Junction from the Varkud Satellite Ribozyme and Identification of Magnesium-Binding Sites Using Paramagnetic Relaxation Enhancement
Nuclear Magnetic Resonance Structure of the III-IV-V Three-Way Junction from the Varkud Satellite Ribozyme and Identification of Magnesium-Binding Sites Using Paramagnetic Relaxation Enhancement
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DOI：
10.1021/bi500826n
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发表时间：
2014-10-07
期刊：
影响因子：
2.9
通讯作者：
Legault, Pascale
中科院分区：
文献类型：
--
作者：
Bonneau, Eric;Legault, Pascale
VS核酶是在脉孢菌的一些天然分离物中发现的催化RNA，其被用作模型系统以提高我们对RNA结构、催化和工程的理解。催化结构域包含五个螺旋结构域（SLII-SLVI），由两个三通接头组织。III-IV-V结是通过与SLV形成吻环相互作用而高亲和力结合底物结构域（SLI）所必需的。在这里，我们确定了高分辨率核磁共振（NMR）结构的47个核苷酸的RNA含有III-IV-V结（J345）。J345 RNA采用典型的C家族三向连接的Y形折叠，茎III和IV之间具有同轴堆叠，茎III和V之间具有锐角。NMR结构揭示了III-IV-V连接的核心包含四个堆叠的碱基三联体、U形转弯基序、交叉链堆叠相互作用、A-次要相互作用和核糖拉链。此外，NMR结构表明，用于稳定茎IV的cCUUGg四环采用了一种新的RNA四环折叠，不同于已知的gCUUGc四环结构。使用Mn 2+诱导的顺磁弛豫增强，我们确定了6个Mg 2+结合位点内J345，包括一个与cCUUGg四环和两个与结核心。J345的NMR结构可能代表活性VS核酶中III-IV-V结的构象，并表明该结作为动态铰链起作用，有助于底物识别和催化。此外，这项研究强调了一个新的作用，家庭C三路交界处的远程三级相互作用。
The VS ribozyme is a catalytic RNA found within some natural isolates of Neurospora that is being used as a model system to improve our understanding of RNA structure, catalysis, and engineering. The catalytic domain contains five helical domains (SLII-SLVI) that are organized by two three-way junctions. The III-IV-V junction is required for high-affinity binding of the substrate domain (SLI) through formation of a kissing loop interaction with SLV. Here, we determine the high-resolution nuclear magnetic resonance (NMR) structure of a 47-nucleotide RNA containing the III-IV-V junction (J345). The J345 RNA adopts a Y-shaped fold typical of the family C three-way junctions, with coaxial stacking between stems III and IV and an acute angle between stems III and V. The NMR structure reveals that the core of the III-IV-V junction contains four stacked base triples, a U-turn motif, a cross-strand stacking interaction, an A-minor interaction, and a ribose zipper. In addition, the NMR structure shows that the cCUUGg tetraloop used to stabilize stem IV adopts a novel RNA tetraloop fold, different from the known gCUUGc tetraloop structure. Using Mn2+-induced paramagnetic relaxation enhancement, we identify six Mg2+-binding sites within J345, including one associated with the cCUUGg tetraloop and two with the junction core. The NMR structure of J345 likely represents the conformation of the III-IV-V junction in the context of the active VS ribozyme and suggests that this junction functions as a dynamic hinge that contributes to substrate recognition and catalysis. Moreover, this study highlights a new role for family C three-way junctions in long-range tertiary interactions.