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In vitro assembly and proteomic analysis of RNA polymerase II complexes
- Joo, Yoo Jin, Ficarro, Scott B., Marto, Jarrod A., Buratowski, Stephen
- Methods 2019
- DNA, DNA-directed RNA polymerase, chromatin, histones, mass spectrometry, messenger RNA, proteomics, screening, transcription (genetics)
- The RNA polymerase II (RNApII) transcription cycle consists of multiple steps involving dozens of protein factors. Here we describe a useful approach to study the dynamics of initiation and early elongation, comprising an in vitro transcription system in which complexes are assembled on immobilized DNA templates and analyzed by quantitative mass spectrometry. This unbiased screening system allows quantitation of RNApII complex components on either naked DNA or chromatin templates. In addition to transcription, the system reproduces co-transcriptional mRNA capping and multiple transcription-related histone modifications. In combination with other biochemical and genetic methods, this approach can provide insights into the mechanistic details of gene expression by RNApII.