Mimicking Co-Transcriptional RNA Folding Using a Superhelicase
Mimicking Co-Transcriptional RNA Folding Using a Superhelicase
复制标题
DOI：
10.1021/jacs.8b03784
复制
发表时间：
2018-08-15
影响因子：
15
通讯作者：
Ha, Taekjip
中科院分区：
文献类型：
--
作者：
Hua, Boyang;Panja, Subrata;Ha, Taekjip
RNA在转录过程中的载体折叠可以产生具有不同生化活性的中间体。在这里，我们设计了一个人工最小系统来模拟体外共转录RNA折叠。在这个系统中，一个预合成的RNA分子开始从它的5‘端折叠，因为它被一个工程解旋酶从异双工中释放出来，在3’到5'方向上易位在互补的DNA链上。这种化学稳定的“超解旋酶”Rep X不断地解开数千对DNA碱基对。预合成的RNA使我们能够灵活地将荧光标记定位在RNA上进行单分子荧光共振能量转移分析，并使我们能够研究矢量折叠过程中的实时构象动力学。我们观察到水稻扭扭核酶的初始二级和三级折叠的明显特征。与Mg2+诱导的再折叠相比，初始矢量三级折叠转变发生得更快，但也更容易发生错误折叠，这可能是由于交替二级结构的顺序形成。这种新的分析方法可以应用于研究其他动力学控制过程，如核糖开关控制和rna -蛋白质组装。
Vectorial folding of RNA during transcription can produce intermediates with distinct biochemical activities. Here, we design an artificial minimal system to mimic cotranscriptional RNA folding in vitro. In this system, a presynthesized RNA molecule begins to fold from its 5'-end, as it is released from a heteroduplex by an engineered helicase that translocates on the complementary DNA strand in the 3'-to-5' direction. This chemically stabilized "superhelicase" Rep X processively unwinds thousands of base pairs of DNA. The presynthesized RNA enables us to flexibly position fluorescent labels on the RNA for single-molecule fluorescence resonance energy transfer analysis and allows us to study real-time conformational dynamics during the vectorial folding process. We observed distinct signatures of the maiden secondary and tertiary folding of the Oryza sativa twister ribozyme. The maiden vectorial tertiary folding transitions occurred faster than Mg2+-induced refolding, but were also more prone to misfolding, likely due to sequential formation of alternative secondary structures. This novel assay can be applied to studying other kinetically controlled processes, such as riboswitch control and RNA-protein assembly.