Model of the RNA polymerase II preinitiation complex containing Pol II and general transcription factors TFIIB, TFIIF, and TFIIE and the function of conserved TFIIE structured domains in transcription initiation

预起始复合体(PIC)的形成是RNA聚合酶(Pol)II转录启动的关键步骤，但大多数导致转录活性复合体形成的事件尚不清楚。我初步的FeBABE切割实验数据表明，通用转录因子(GTF)TFIIE在PIC中有一个意想不到的位置。我发现TFIIE大亚基Tfa1的保守的N末端翼状螺旋结构域(WH-D)位于Pol II叶之上，位于Pol II与TFIIF的两个结构域相互作用的界面上。TFIIE、TFIIF和POL II之间的相互作用可能会将TFIIE锚定在PIC中，修改TFIIF-POL II相互作用，并允许TFIIE与上游DNA相互作用。我还发现，在PIC中，保守的Tfa1锌结合结构域(ZB-D)位于Pol II裂隙中，靠近TFIIB的活性部位和B-阅读器区域，从那里它可能与Pol II、TFIIB和TFIIF Tfg2连接器域的灵活区域相互作用，促进起始和转录起始位点的选择。为了回答TFIIE如何在功能上影响这些元件，并直接和/或间接促进转录启动，将通过遗传分析和生化分析来分析TFIIE Tfa1 ZB-D和WH-D在转录启动中的功能贡献。我将进一步扩展TFIIE与POL II和GTF如TFIIF和TFIIB相互作用的初步映射数据。为了最终确定TFIIE在PIC中的位置，还需要进一步的FeBABE切割实验，将FeBABE与TFIIE其他结构区域表面的残基连接起来。综上所述，该项目将使我们能够为POL II PIC创建迄今最详细的模型，其中包含POL II以及GTF TFIIB、TFIIF和TFIIE，从而极大地有助于了解PIC的完整结构组成以及导致开放复合体形成和转录启动的机制步骤。

The formation of the preinitiation complex (PIC) is a key step in RNA polymerase (Pol) II transcription initiation, but most of the events leading to the formation of a transcription competent complex are unknown. Data from my preliminary FeBABE cleavage experiments suggest an unexpected location of the general transcription factor (GTF) TFIIE in PIC. I find that the conserved N-terminal winged helix domain (WH-D) of the TFIIE large subunit Tfa1 is located above the Pol II lobe between the Pol II interaction interfaces with the two structured domains of TFIIF. Interactions between TFIIE, TFIIF, and Pol II potentially anchor TFIIE in the PIC, modify TFIIF-Pol II interactions, and allow TFIIE to interact with upstream DNA. I also find that in the PIC, the conserved Tfa1 zinc binding domain (ZB-D) is positioned in the Pol II cleft, close to the active site and B-reader region of TFIIB, from where it may interact with flexible regions of Pol II, TFIIB, and the TFIIF Tfg2 linker domain to promote initiation and transcription start site selection. To answer how TFIIE functionally influences these elements, and directly and/or indirectly promotes transcription initiation, the functional contribution of the TFIIE Tfa1 ZB-D and WH-D in transcription initiation will be analyzed by a genetic assay and biochemical assays. I will further extend the preliminary mapping data of TFIIE interactions with Pol II and GTFs like TFIIF and TFIIB. To conclusively position TFIIE in the PIC, further FeBABE cleavage experiments with FeBABE linked to residues on the surface of other structured regions of TFIIE will also be required. Taken together, this project will enable us to create the most detailed model for the Pol II PIC so far, containing Pol II and the GTFs TFIIB, TFIIF, and TFIIE, thereby greatly contributing to the understanding of the complete structural composition of the PIC and the mechanistic steps leading to formation of the open complex and transcription initiation.