Automated RNA Structure Prediction Uncovers a Kink-Turn Linker in Double Glycine Riboswitches
Automated RNA Structure Prediction Uncovers a Kink-Turn Linker in Double Glycine Riboswitches
复制标题
DOI：
10.1021/ja2093508
复制
发表时间：
2012-01-25
影响因子：
15
通讯作者：
Das, Rhiju
中科院分区：
文献类型：
--
作者：
Kladwang, Wipapat;Chou, Fang-Chieh;Das, Rhiju
功能性RNA分子的三级结构仍然难以破译。新一代自动化 RNA 结构预测方法可能有助于解决这些挑战，但尚未经过实验验证。在这里，我们将四种预测工具应用于一类可以协同结合两个配体的双甘氨酸核糖开关。一种新方法 (BPPalign)、RMdetect、JAR3D 和 Rosetta 3D 建模对新茎 P0 和扭结转向基序给出了一致的预测。这些元件构成了 RNA 双适体之间的连接体。使用 N-甲基靛红酸酐、硫酸二甲酯和 1-环己基-3-(2-吗啉乙基)碳二亚胺甲对甲苯磺酸盐对具核梭杆菌核糖开关进行化学作图、突变和图谱研究以及突变/拯救实验都为结构接头提供了强有力的证据。在允许严格热力学分析的溶液条件下，破坏这种螺旋-连接-螺旋结构会导致 RNA 的两个甘氨酸结合转变的解离常数降低 120 倍和 6-30 倍，相当于 4.3 +/- 0.5 kcal/mol 的总体能量影响。由于 RNA 过度截短，之前的生化和晶体学研究并未包括这一关键元素。我们推测，该RNA和其他功能性RNA的侧翼区域中可能存在一些其他未被发现的元素，并且自动预测工具可以在它们的检测和解剖中发挥有用的作用。
The tertiary structures of functional RNA molecules remain difficult to decipher. A new generation of automated RNA structure prediction methods may help address these challenges but have not yet been experimentally validated. Here we apply four prediction tools to a class of double glycine riboswitches that can bind two ligands cooperatively. A novel method (BPPalign), RMdetect, JAR3D, and Rosetta 3D modeling give consistent predictions for a new stem P0 and a kink-turn motif. These elements structure the linker between the RNAs' double aptamers. Chemical mapping on the Fusobacterium nucleatum riboswitch with N-methylisatoic anhydride, dimethyl sulfate and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate probing, mutate-and-map studies, and mutation/rescue experiments all provide strong evidence for the structured linker. Under solution conditions that permit rigorous thermodynamic analysis, disrupting this helix-junction-helix structure gives 120- and 6-30-fold poorer dissociation constants for the RNA's two glycine-binding transitions, corresponding to an overall energetic impact of 4.3 +/- 0.5 kcal/mol. Prior biochemical and crystallography studies did not include this critical element due to over-truncation of the RNA. We speculate that several further undiscovered elements are likely to exist in the flanking regions of this and other functional RNAs, and automated prediction tools can play a useful role in their detection and dissection.