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

前起始复合物（preinitiation complex，PIC）的形成是RNA聚合酶II（polymerase，Pol）转录起始的关键步骤，但大多数导致转录复合物形成的事件尚不清楚。从我的初步FeBABE切割实验的数据表明，一个意想不到的位置的一般转录因子（GTF）TFIIE在PIC。我发现TFIIE大亚基Tfa 1保守的N-末端翼螺旋结构域（WH-D）位于TFIIF的两个结构域的Pol II相互作用界面之间的Pol II叶上方。TFIIE、TFIIF和Pol II之间的相互作用潜在地将TFIIE锚在PIC中，修饰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Tfa 1锌结合结构域（ZB-D）位于Pol II裂缝中，靠近TFIIB的活性位点和B-阅读区，从那里它可以与Pol II，TFIIB和TFIIF Tfg 2连接结构域的柔性区域相互作用，以促进起始和转录起始位点选择。为了回答TFIIE如何在功能上影响这些元件，并直接和/或间接促进转录起始，将通过遗传测定和生物化学测定来分析TFIIE Tfa 1 ZB-D和WH-D在转录起始中的功能贡献。我将进一步扩展TFIIE与Pol II和TFIIF和TFIIB等GTF相互作用的初步映射数据。为了最终将TFIIE定位在PIC中，还需要进一步的FeBABE切割实验，其中FeBABE连接到TFIIE的其他结构化区域的表面上的残基。总之，该项目将使我们能够创建迄今为止最详细的Pol II PIC模型，包含Pol II和GTF TFIIB，TFIIF和TFIIE，从而大大有助于理解PIC的完整结构组成以及导致形成开放复合物和转录起始的机械步骤。

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.