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.

中文翻译：

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单分子 FRET 方法研究 RNA 聚合酶 II 通过核小体的转录延伸动力学


通过核小体的转录延伸是一种精确协调的活动，以确保 RNA 的及时产生和共转录组蛋白修饰的准确调节。核小体积极参与各个水平的转录调控，并在转录延伸过程中对 RNA 聚合酶 II (RNAPII) 施加物理屏障。尽管具有重要意义，但关于 RNAPII 在转录延伸过程中如何沿着核小体 DNA 易位以及核小体结构如何动态地适应 RNAPII 进展所需的变化的详细动态仍然知之甚少。通过核小体的转录延伸是一个复杂的过程，通过整体测量来研究该过程中核小体结构的变化是令人畏惧的。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延伸复合物同步到指定位置以获得所需的样品均匀性。在此我们回顾一下我们最近开发的单分子FRET实验系统和方法，该系统和方法弥补了这一缺陷。利用我们的方法，人们可以追踪转录延伸过程中单个核小体结构的变化。我们证明，该方法能够详细测量通过核小体的转录延伸动力学及其转录因子的调节，这可以很容易地扩展到环境变量和组蛋白翻译后修饰在调节转录延伸中的作用的研究。