RNA Polymerase II Holoenzyme

RNA聚合酶II （Pol II）催化所有蛋白编码基因的表达。数据表明，基因转录是由Pol II酶的一种形式调控的，称为Pol II全酶，它是核心Pol II与基础转录因子和共激活因子子集结合的巨大复合物。协同激活因子是一种蛋白质，它介导从增强结合调节蛋白到基础机制的信号。转录调节因子的生物化学进展揭示了基础转录因子如何发挥作用，但对增强子结合调节因子如何通过共激活子与基础机制进行通信知之甚少。本项目的重点是确定共激活剂和调节修饰影响pol II全酶功能的机制。我们已经发现DNA拓扑异构酶II α作为Pol II全酶的共激活剂。虽然这种酶在基因组的复制中起着至关重要的作用，但其转录功能尚未得到证实。我们发现DNA拓扑异构酶II通过染色质依赖的共激活子活性在转录中起作用。该项目的第一个目标是确定DNA拓扑异构酶iiialpha调节染色质模板转录的机制，并鉴定其表达受拓扑异构酶iiialpha调节的基因。该项目的第二个目标是开发一种可靠的Pol II全酶纯化方法，以表征其多肽组成，对可能影响功能的特定亚基的修饰，并描述Pol II全酶是否存在亚群。总之，这些目标将定义细胞中这个关键的mRNA合成机器，确定修饰和亚群如何可能产生具有特殊功能的Pol II全酶复合物，并阐明拓扑异构酶II如何调节转录。

RNA polymerase II (Pol II) catalyzes the expression of all protein- encoding genes. Data suggest that gene transcription is regulated by a form of the Pol II enzyme known as the Pol II holoenzyme, which is a massive complex of the core Pol II bound to a subset of basal transcription factors and co-activators. A co-activator is a protein that mediates the signal from enhance binding regulatory proteins to the basal machinery. Advances in the biochemistry of transcription regulators has revealed much about how the basal transcription factors function, but little is known about how the enhancer bound regulators communicate with the basal machinery through the co-activators. The focus of this project is to identify mechanisms by which co-activators and regulated modifications influence pol II holoenzyme function. We have found that DNA topoisomerase IIalpha acts as a co-activator in the Pol II holoenzyme. While this enzyme has been known to be critical in the replication of the genome, its transcription function has not been demonstrated We found that DNA topoisomerase II functions in transcription by a chromatin-dependent co-activator activity. The first aim of this project will identify the mechanism by which DNA topoisomerase IIALPHA regulates transcription on chromatin templates and identify genes whose expression is regulated by topoisomerase IIalpha. The second aim of this project will develop a reliable purification of the Pol II holoenzyme in order to characterize its polypeptide composition, modifications to specific subunits which may affect function, and delineate whether subpopulations exist of the Pol II holoenzyme. Together, these aims will define this critical mRNA synthesizing machine of the cell, identify how modifications and subpopulations perhaps create Pol II holoenzyme complexes with specialized function and elucidate how topoisomerase II regulates transcription.