Division of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, United States.
Division of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, United States; Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, United States.
Methods. 2019 Apr 15;159-160:29-34. doi: 10.1016/j.ymeth.2019.02.019. Epub 2019 Feb 22.
Transcription, catalyzed by RNA polymerase II (Pol II) in eukaryotes, is the first step in gene expression. RNA Pol II is a 12-subunit enzyme complex regulated by many different transcription factors during transcription initiation, elongation, and termination. During elongation, Pol II encounters various types of obstacles that can cause transcriptional pausing and arrest. Through decades of research on transcriptional pausing, it is widely known that Pol II can distinguish between different types of obstacles by its active site. A major class of obstacles is DNA lesions. While some DNA lesions can cause transient transcriptional pausing, which can be bypassed by Pol II itself or with the help from other elongation factors, bulky DNA damage can cause prolonged transcriptional pausing and arrest, which signals for transcription coupled repair. Using biochemical and structural biology approaches, the outcomes of many different types of DNA lesions, DNA modifications, and DNA binding molecules to transcription were studied. In this mini review, we will describe the in vitro transcription assays with Pol II to investigate the impacts of various DNA lesions on transcriptional outcomes and the crystallization method of lesion-arrested Pol II complex. These methods can provide a general platform for the structural and biochemical analysis of Pol II transcriptional pausing and bypass mechanisms.
转录是基因表达的第一步，在真核生物中由 RNA 聚合酶 II（Pol II）催化。RNA Pol II 是一种由 12 个亚基组成的酶复合物，在转录起始、延伸和终止过程中受到许多不同转录因子的调节。在延伸过程中，Pol II 遇到各种类型的障碍，这些障碍可能导致转录暂停和停滞。通过几十年对转录暂停的研究，人们普遍认为 Pol II 可以通过其活性位点来区分不同类型的障碍。一类主要的障碍是 DNA 损伤。虽然一些 DNA 损伤会导致短暂的转录暂停，Pol II 本身或其他延伸因子可以绕过这种暂停，但大体积的 DNA 损伤会导致转录的长时间暂停和停滞，从而引发转录偶联修复。利用生化和结构生物学方法，研究了许多不同类型的 DNA 损伤、DNA 修饰和 DNA 结合分子对转录的影响。在这篇综述中，我们将描述使用 Pol II 的体外转录实验，以研究各种 DNA 损伤对转录结果的影响，以及损伤停滞的 Pol II 复合物的结晶方法。这些方法可以为 Pol II 转录暂停和绕过机制的结构和生化分析提供一个通用平台。
