Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization
Monitoring transcriptional activity by RNA polymerase II in vitro using single molecule co-localization
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DOI：
10.1016/j.ymeth.2019.03.006
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发表时间：
2019-04-15
期刊：
影响因子：
4.8
通讯作者：
Kugel, Jennifer F.
中科院分区：
文献类型：
--
作者：
Ly, Elina;Goodrich, James A.;Kugel, Jennifer F.
RNA聚合酶II（Pol II）转录真核生物mRNA基因。为了启动转录，在靶基因的核心启动子上形成含有Pol II和一般转录因子（GTF）的前起始复合物（PIC）。在细胞中，这一过程受转录激活因子、共激活因子和染色质修饰复合物的调节。重组体外转录系统是研究酶学和转录反应基本步骤的重要工具。在这些系统中，由于在启动子处组装的转录活性和非活性复合物的异质混合物，研究转录可能是复杂的。因此，我们开发了一种技术，使用单分子显微镜来解决这种异质性，并区分转录活性复合物从非活性复合物。该系统使用荧光标记的启动子DNA和由纯化的人Pol II和GTF组成的最小重建转录系统。在这里，我们描述的材料，方法和分析需要在单分子水平上研究Pol II转录。我们的单分子方法的灵活性允许适应，以回答不同的转录机制的问题，否则将难以研究使用合奏测定。
RNA polymerase II (Pol II) transcribes eukaryotic mRNA genes. To initiate transcription, pre-initiation complexes (PICs) containing Pol II and general transcription factors (GTFs) form on the core promoters of target genes. In cells this process is regulated by transcriptional activators, co-activators, and chromatin modifying complexes. Reconstituted in vitro transcription systems are important tools for studying the enzymology and fundamental steps in the transcription reaction. In these systems, studying transcription can be complex due to the heterogeneous mixture of transcriptionally active and inactive complexes that assemble at promoters. Accordingly, we developed a technique to use single molecule microscopy to resolve this heterogeneity and distinguish transcriptionally active complexes from inactive complexes. This system uses fluorescently-labeled promoter DNA and a minimal reconstituted transcription system consisting of purified human Pol II and GTFs. Here we describe the materials, methods, and analysis required to study Pol II transcription at the single molecule level. The flexibility of our single molecule method allows for adaptation to answer diverse mechanistic questions about transcription that would otherwise be difficult to study using ensemble assays.