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

在真核生物中，RNA聚合酶II （Pol II）对基因的转录是基因表达的第一步。Pol II在五种一般转录因子的帮助下开始转录。一般转录因子TFIIE和TFIIH在最后一步参与起始前复合物（PIC），通过稳定和激活PIC开始转录，并参与PIC的解离和延伸复合物的形成。TFIIE由两个亚基TFIIE α和TFIIE β组成，形成了一个四聚体的alpha2beta2形式。每个亚基都可以在大肠杆菌中独立表达并溶解。因此，我们构建了tfiieβ的缺失突变，在大肠杆菌中表达，纯化并用于鉴定基础转录必需区域、tfiieα结合区域以及另一种通用转录因子TFIIB和tfiifβ的结合区域，并证明这些区域实际上对刺激tfiih衍生的ctd激酶活性很重要。在TFIIH的情况下，它表现出基础的转录活性，但当比较纯化的TFIIH和CAK对转录的参与时，由TFIIH的三个亚基组成的细胞周期调节因子CAK不能取代这种转录活性。然后，比较TFIIH和CAK激酶活性的特异性，发现TFIIH磷酸化Pol II的CTD，但不磷酸化周期蛋白依赖性激酶CDK4，而CAK不能磷酸化CTD，但磷酸化CDK4。我们目前正在通过纯化这些突变体，研究这些因子对细胞周期的参与，以及具有XPB或XPD缺陷的突变体TFIIH对转录和细胞周期调控的影响。总之，我们可以通过激酶活性证明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.