Multiple metal-binding cores are required for metalloregulation by M-box riboswitch RNAs.
Multiple metal-binding cores are required for metalloregulation by M-box riboswitch RNAs.
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M-box 核糖开关 RNA 的金属调节需要多个金属结合核心。
DOI：
10.1016/j.jmb.2009.07.033
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发表时间：
2009
影响因子：
5.6
通讯作者：
Winkler,WadeC
中科院分区：
文献类型：
--
作者：
Wakeman,CatherineA;Ramesh,Arati;Winkler,WadeC
核糖开关是调节性 RNA，可响应与细胞内代谢物或金属的直接关联来控制下游基因表达。通常，核糖开关适体结构域与单个小分子代谢物结合。相比之下，M-box 核糖开关适体的 X 射线晶体结构模型显示不存在有机代谢物配体，但存在至少六个紧密相连的镁。这一观察结果与 M-box 核糖开关作为细胞内镁传感器的作用非常吻合，尽管为了这些目的无疑还需要额外的非特异性金属相互作用。为了更深入地了解 M-box RNA 的金属调节能力，我们试图确定核糖开关功能是否需要全部或部分 RNA 螯合镁离子。为了完成这项任务，通过随机掺入硫代磷酸酯核苷酸类似物，同时但单独地扰动每个镁结合位点，并研究了 RNA 分子在不同镁水平下折叠的能力。这些数据表明，结构模型中观察到的所有镁离子对于镁依赖性三级结构的形成都很重要。此外，这些功能数据揭示了潜在金属结合位点的新核心，这些位点可能有助于关键三级相互作用的形成，并且之前在结构模型中未观察到。从这些数据可以清楚地看出，M-box RNA 需要金属离子网络的特异性结合才能部分实现其金属调节功能。
Riboswitches are regulatory RNAs that control downstream gene expression in response to direct association with intracellular metabolites or metals. Typically, riboswitch aptamer domains bind to a single small-molecule metabolite. In contrast, an X-ray crystallographic structural model for the M-box riboswitch aptamer revealed the absence of an organic metabolite ligand but the presence of at least six tightly associated magnesiums. This observation agrees well with the proposed role of the M-box riboswitch in functioning as a sensor of intracellular magnesium, although additional nonspecific metal interactions are also undoubtedly required for these purposes. To gain greater functional insight into the metalloregulatory capabilities of M-box RNAs, we sought to determine whether all or a subset of the RNA-chelated magnesium ions were required for riboswitch function. To accomplish this task, each magnesium-binding site was simultaneously yet individually perturbed through random incorporation of phosphorothioate nucleotide analogues, and RNA molecules were investigated for their ability to fold in varying levels of magnesium. These data revealed that all of the magnesium ions observed in the structural model are important for magnesium-dependent tertiary structure formation. Additionally, these functional data revealed a new core of potential metal-binding sites that are likely to assist formation of key tertiary interactions and were previously unobserved in the structural model. It is clear from these data that M-box RNAs require specific binding of a network of metal ions for partial fulfillment of their metalloregulatory functions.