|Budget Amount *help
¥7,600,000 (Direct Cost : ¥7,600,000)
Fiscal Year 1997 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1996 : ¥5,700,000 (Direct Cost : ¥5,700,000)
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.