Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II
Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II
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DOI：
10.1016/j.ymeth.2019.01.009
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发表时间：
2019-04-15
期刊：
影响因子：
4.8
通讯作者：
Lee, Tae-Hee
中科院分区：
文献类型：
--
作者：
Lee, Jaehyoun;Crickard, J. Brooks;Lee, Tae-Hee
通过核小体的转录延伸是一种精确的协调活动，以确保RNA的及时产生和共转录的组蛋白修饰的准确调节。核小体在不同水平上积极参与转录调控，并在转录延伸过程中对RNA聚合酶II(RNAPII)施加物理障碍。尽管RNAPII具有很高的意义，但关于RNAPII在转录延伸过程中如何沿核小体DNA移位以及核小体结构如何动态适应RNAPII进展所需的变化的详细动力学仍不清楚。通过核小体进行转录伸长是一个复杂的过程，通过系综测量来研究这一过程中核小体结构的变化是令人望而生畏的。这是因为几乎不可能以足够高的效率将核小体或亚核小体内的伸长复合体同步到指定的位置，以达到所需的样品均一性。在这里，我们回顾了我们最近开发的单分子FRET实验系统和方法，弥补了这一不足。使用我们的方法，我们可以跟踪单个核小体在转录延伸过程中结构的变化。我们证明，这种方法能够详细地测量通过核小体的转录伸长动力学以及转录因子对转录伸长的调控，可以很容易地扩展到研究环境变量和组蛋白翻译后修饰在调控转录伸长中的作用。
Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Transcription elongation through the nucleosome is a complex process and investigating the changes of the nucleosome structure during this process by ensemble measurements is daunting. This is because it is nearly impossible to synchronize elongation complexes within a nucleosome or a sub-nucleosome to a designated location at a high enough efficiency for desired sample homogeneity. Here we review our recently developed single-molecule FRET experimental system and method that has fulfilled this deficiency. With our method, one can follow the changes in the structure of individual nucleosomes during transcription elongation. We demonstrated that this method enables the detailed measurements of the kinetics of transcription elongation through the nucleosome and its regulation by a transcription factor, which can be easily extended to investigations of the roles of environmental variables and histone post-translational modifications in regulating transcription elongation.