Wakeman Catherine A, Ramesh Arati, Winkler Wade C
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, 75390, USA.
J Mol Biol. 2009 Sep 25;392(3):723-35. doi: 10.1016/j.jmb.2009.07.033. Epub 2009 Jul 17.
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.
核糖开关是一类调控性RNA，可通过与细胞内代谢物或金属直接结合来控制下游基因表达。通常，核糖开关适体结构域会与单一小分子代谢物结合。相比之下，M盒核糖开关适体的X射线晶体学结构模型显示，其中不存在有机代谢物配体，但存在至少六个紧密结合的镁离子。这一观察结果与M盒核糖开关作为细胞内镁离子传感器的作用十分相符，尽管毫无疑问，这些作用还需要额外的非特异性金属相互作用。为了更深入了解M盒RNA的金属离子调控能力，我们试图确定核糖开关功能是否需要所有或部分与RNA螯合的镁离子。为完成这项任务，通过随机掺入硫代磷酸核苷酸类似物，对每个镁离子结合位点进行同时但单独的扰动，并研究RNA分子在不同镁离子水平下折叠的能力。这些数据表明，结构模型中观察到的所有镁离子对于依赖镁离子的三级结构形成都很重要。此外，这些功能数据还揭示了一个新的潜在金属结合位点核心，它们可能有助于形成关键的三级相互作用，而这些位点在结构模型中此前并未观察到。从这些数据可以清楚地看出，M盒RNA需要特定的金属离子网络结合，才能部分实现其金属离子调控功能。
