During transcription along nucleosomal DNA, RNA polymerase II (Pol II) pauses at multiple positions and induces formation of multiple intermediates that aid in maintaining proper chromatin structure. To describe the kinetics of this multiple-step reaction, we utilized a computational model-based approach and KinTek Explorer software to analyze the time courses. Here we describe the stepwise protocol for analysis of the kinetics of transcription through a nucleosome that provides the rate constants for each step of this complex process. We also present an example where this time-resolved approach was applied to study the mechanism of histone chaperone FACT action during Pol II transcription through a single nucleosome by comparing the rate constants derived in the presence or in the absence of FACT.

中文翻译：

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染色质转录的时间分辨分析

在沿核小体 DNA 转录期间，RNA 聚合酶 II (Pol II) 在多个位置暂停并诱导形成多个中间体，这些中间体有助于维持正确的染色质结构。为了描述这种多步反应的动力学，我们利用基于计算模型的方法和 KinTek Explorer 软件来分析时间进程。在这里，我们描述了通过核小体分析转录动力学的逐步协议，该核小体为这个复杂过程的每个步骤提供速率常数。我们还提供了一个例子，其中通过比较在存在或不存在 FACT 的情况下得出的速率常数，应用这种时间分辨方法来研究组蛋白伴侣 FACT 在通过单个核小体转录期间组蛋白伴侣 FACT 作用的机制。