Study of general transcription factors TFIIE and TFIIH on the effect to multistep transcriptional regulation and cell cycle

在真核生物中，通过RNA聚合酶II（Pol II）的基因转录是基因表达的第一步。Pol II在五种通用转录因子的协助下开始转录。通用转录因子TFIIE和TFIIH在最后一步参与前起始复合物（PIC），通过稳定和激活PIC启动转录，并参与PIC的解离和在向延伸过渡时形成延伸复合物。TFIIE由两个亚基TFIIE α和TFIIE β组成，并形成α 2 β 2形式的四聚体。每个亚基可以在大肠杆菌中独立表达并变得可溶。因此，构建TFIIE β的缺失突变，在大肠杆菌中表达，纯化并用于鉴定基础转录的必需区域、TFIIE α结合区域和另一种通用转录因子TFIIB和TFIIF β的结合区域，并证明这些区域对于刺激TFIIH衍生的CTD-激酶活性实际上是重要的。当比较纯化的TFIIH和CAK参与转录时，其表现出基本的转录活性，但是由TFIIH的三个亚基组成的细胞周期调节因子CAK不能替代该转录活性。然后，比较TFIIH和CAK的激酶活性的特异性，发现TFIIH磷酸化Pol II的CTD，但不磷酸化细胞周期蛋白依赖性激酶CDK 4，另一方面，CAK不能磷酸化CTD，但磷酸化CDK 4。我们目前正在研究这些因素在细胞周期中的参与和突变TFIIH的影响，具有缺陷的XPB或XPD，通过纯化这些突变体的转录和细胞周期调控。总之，我们可以证明通过激酶活性的转录和细胞周期的特定调控的重要性。

In eukaryotes, transcription of the genes by RNA polymerase II (Pol II) is the first step of gene expression. Pol II starts transcription with the assistance of five general transcription factors. The general transcription factors TFIIE and TFIIH attend to the preinitiation complex (PIC) at the last step, start transcription by stabilizing and activating the PIC,and are involved in dissociation of the PIC and in formation of the elongation complex at the transition to elongation. TFIIE consists of two subunits TFIIEalpha and TFIIEbeta and forms a tetramer of alpha2beta2 form. Each subunit can be expressed independently in E.coli and becomes soluble. Therefore, the deletion mutations of TFIIEbeta were constructed, expressed in E.coli, purified and used to identify the essential region for basal transcription, the TFIIEalpha binding region and the binding region of another general transcription factors TFIIB and TFIIFbeta, and to demonstrate that these regions are actually important for the stimulation of TFIIH-derived CTD-kinase activity.In case of TFIIH,this exhibited basal transcription activity but the cell cycle regulating factor CAK consisting of three subunits of TFIIH could not replace this transcription activity when the involvement in transcription of purified TFIIH and CAK was compared. Then, the specificities of kinase activities of TFIIH and CAK were compared and it was found that TFIIH phosphorylated the CTD of Pol II but did not phosphorylate the cyclin-dependent kinase CDK4 and, on the other hand, CAK could not phosphorylate the CTD but did phosphorylate CDK4. We are currently studying the involvement of these factors in cell cycle and the effects of the mutant TFIIH,that possess defects in either XPB or XPD,on transcription and cell cycle regulation by purifying those mutants. In summary, we could demonstrate importance of specific regulations of both transcription and cell cycle through kinase activities.