A single-molecule study of RNA catalysis and folding
A single-molecule study of RNA catalysis and folding
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DOI：
10.1126/science.288.5473.2048
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发表时间：
2000-06-16
期刊：
影响因子：
56.9
通讯作者：
Chu, S
中科院分区：
文献类型：
--
作者：
Zhuang, XW;Bartley, LE;Chu, S
利用荧光显微镜，我们研究了单个四膜虫嗜热核酶分子的催化和折叠。所使用的染料标记的和表面固定的核酶被证明是功能上无法区分的未修饰的游离核酶在溶液中。在单分子时间轨迹中直接观察到可逆的局部折叠步骤，其中双链体从核酶核心对接和脱离，从而允许确定速率常数和表征过渡态。观察到很少填充的对接状态，无法通过系综方法测量。在整个折叠过程中，观察到中间折叠状态和多个折叠途径。除了观察先前建立的折叠途径外，还发现了一种观察到的折叠速率常数为每秒1次的途径。这些结果确立了单分子荧光作为检查RNA折叠的有力工具。
Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible Local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state, A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.