Direct observation of hierarchical folding in single riboswitch aptamers
Direct observation of hierarchical folding in single riboswitch aptamers
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DOI：
10.1126/science.1151298
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发表时间：
2008-02-01
期刊：
影响因子：
56.9
通讯作者：
Block, Steven M.
中科院分区：
文献类型：
--
作者：
Greenleaf, William J.;Frieda, Kirsten L.;Block, Steven M.
核开关通过改变配体结合RNA适体的结构来调节基因。利用基于RNA聚合酶分子原位转录的光学捕获实验，单个含有pbuE腺嘌呤核糖开关适配体的新生RNA被展开并重新折叠。通过测量分子轮廓长度、动力学和含腺嘌呤和不含腺嘌呤的能量学，用力延伸曲线和恒力作用下的折叠轨迹来表征多种折叠状态。不同的折叠步骤与关键的二级或三级结构的形成和配体结合有关。腺嘌呤诱导的适体中最弱螺旋的稳定化，即调节作用的机械开关，被直接观察到。这些结果提供了适体中分层折叠的综合视图，展示了复杂折叠如何被分解成组成部分，并为三级结构的形成提供了进一步的见解。
Riboswitches regulate genes through structural changes in ligand- binding RNA aptamers. With the use of an optical- trapping assay based on in situ transcription by a molecule of RNA polymerase, single nascent RNAs containing pbuE adenine riboswitch aptamers were unfolded and refolded. Multiple folding states were characterized by means of both force- extension curves and folding trajectories under constant force by measuring the molecular contour length, kinetics, and energetics with and without adenine. Distinct folding steps correlated with the formation of key secondary or tertiary structures and with ligand binding. Adenine- induced stabilization of the weakest helix in the aptamer, the mechanical switch underlying regulatory action, was observed directly. These results provide an integrated view of hierarchical folding in an aptamer, demonstrating how complex folding can be resolved into constituent parts, and supply further insights into tertiary structure formation.