Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Protein Analysis and Proteomics Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Methods. 2019 Apr 15;159-160:146-156. doi: 10.1016/j.ymeth.2019.02.005. Epub 2019 Feb 13.
Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.
RNA 聚合酶 II（RNAPII）的转录过程被大量的翻译后修饰所修饰，这些修饰标志着转录的不同阶段。一种重要的修饰是 RNAPII 的泛素化，它发生在对许多不同刺激的反应中，这些刺激会导致 RNAPII 停滞，如 DNA 损伤剂、RNAPII 抑制剂或核苷酸池的耗尽。停滞的 RNAPII 触发了所谓的“最后手段途径”，其中涉及 RNAPII 的多泛素化和蛋白酶体介导的降解。已经描述了不同的方法来研究 RNAPII 的多泛素化和降解，每种方法都有其自身的优点和缺点。在这里，我们描述了优化的检测泛素化 RNAPII 和研究其降解的策略，但这些方案也适用于研究其他泛素化蛋白。
