Analysis of RNA polymerase-promoter complex formation.
Analysis of RNA polymerase-promoter complex formation.
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RNA 聚合酶-启动子复合物形成的分析。
DOI：
10.1016/j.ymeth.2008.10.018
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Gourse,RichardL
中科院分区：
文献类型：
--
作者：
Ross,Wilma;Gourse,RichardL
细菌启动子的鉴定和表征并不像人们想象的那么简单。启动子在与共有识别元件序列的相似性、活性以及转录因子的利用方面差异很大，并且通常必须使用多种方法来提供理解启动子调控的框架。在缺乏额外调节因子（基础启动子功能）的情况下，对 RNA 聚合酶-启动子复合物形成的表征可以为理解营养或环境因素最终靶向的转录起始步骤提供基础。启动子可以在体内使用遗传方法定位，但 RNAP-启动子复合物的详细特性在体外研究最为有效。我们首先描述了体内鉴定细菌启动子和转录起始位点的方法，包括启动子-报告基因融合和引物延伸。然后，我们描述了多种体外表征 RNAP 启动子复合物的方法，包括体外转录、凝胶迁移率变化测定、足迹分析和过滤器结合。利用这些方法不仅可以确定基础启动子强度，还可以确定转录起始复合物形成和衰变的速率。
Bacterial promoter identification and characterization is not as straightforward as one might presume. Promoters vary widely in their similarity to the consensus recognition element sequences, in their activities, and in their utilization of transcription factors, and multiple approaches often must be used to provide a framework for understanding promoter regulation. Characterization of RNA polymerase-promoter complex formation in the absence of additional regulatory factors (basal promoter function) can provide a basis for understanding the steps in transcription initiation that are ultimately targeted by nutritional or environmental factors. Promoters can be localized using genetic approaches in vivo, but the detailed properties of the RNAP-promoter complex are studied most productively in vitro. We first describe approaches for identification of bacterial promoters and transcription start sites in vivo, including promoter-reporter fusions and primer-extension. We then describe a number of methods for characterization of RNAP-promoter complexes in vitro, including in vitro transcription, gel mobility shift assays, footprinting, and filter binding. Utilization of these methods can result in determination of not only basal promoter strength but also the rates of transcription initiation complex formation and decay.