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Time-resolved analysis of transcription through chromatin
- Chang, Han-Wen, Hsieh, Fu-Kai, Patel, Smita S., Studitsky, Vasily M.
- Methods 2019
- DNA, DNA-directed RNA polymerase, computer software, histones, molecular chaperones, nucleosomes, transcription (genetics)
- 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.