Deciphering the phosphorylation pattern of RNA polymerase II for eukaryotic transcription

摘要 最大的RNA聚合酶II亚基的C末端结构域(CTD)是一个独特的CTD序列 (Y1S2P3T4S5P6S7)重复多次，在真核生物中大部分是保守的。此域协调 通过翻译后修饰将转录因子招募到POL II，其中 削弱高效的转录过程，导致细胞死亡。准确的磷酸化状态 通过激酶和磷酸酶对CTD七肽重复序列中的不同残基进行比较对于准确招募 调节转录过程的蛋白质。我们的实验室利用我们在蛋白质化学方面的丰富经验 通过研究磷酸化的活性和特异性，了解转录过程中磷酸化的精确模式 这些激酶和磷酸酶。我们试图了解CTD的翻译后修饰是如何 在生物事件期间发生改变，以及这种变化如何反映在转录结果中。我们用 多学科方法，包括结构生物学、生物化学、质谱学方法学和 用整体转录组分析研究RNA不同修饰状态的分子机制 聚合酶II协调真核转录。 配备了关于磷酸酶功能和活性的广泛知识，我们进一步探索如何 化学抑制SCP1是一种属于HAD超家族的人类磷酸酶，可以抑制一些生长 胶质母细胞瘤细胞。我们利用我们在基于结构的抑制剂设计方面的经验来识别共价和非共价 以这种独特的磷酸酶为靶点的共价抑制剂。

ABSTRACT The C-terminal domain (CTD) of the largest RNA polymerase II subunit is a unique CTD sequence (Y1S2P3T4S5P6S7) repeated many times and is mostly conserved in eukaryotes. This domain coordinates the recruitment of transcriptional factors to Pol II through its post-translational modifications, the loss of which cripples the highly efficient transcription process and causes the cell to die. The accurate phosphorylation state of different residues in the CTD heptad repeats by kinase and phosphatases is crucial to precisely recruiting proteins to mediate the transcription process. Our lab utilizes our extensive experience in protein chemistry to understand the precise pattern of phosphorylation during transcription by investigating the activity and specificity of these kinases and phosphatases. We seek to understand how the post-translational modifications of CTD are altered during biological events and how such changes are reflected in the outcome of transcription. We use multi-disciplinary methods including structural biology, biochemistry, mass spectrometry methodology, and global transcriptome analysis to investigate the molecular mechanism of how different modification states of RNA polymerase II coordinate the eukaryotic transcription. Equipped with extensive knowledge about the phosphatase function and activity, we further explore how the chemical inhibition of SCP1, a human phosphatase belonging to HAD superfamily, thwarts some of the growth glioblastoma cells. We utilized our experience in structural-based inhibitor design to identify covalent and non- covalent inhibitors targeting this unique phosphatase.