Biochemical and Ganetical Analyses of Organized Regularly Mechanism of Gene Expression by Various Transcription Super-Complex.

1.调解人复合体的特征。该介体由20多个亚基组成，将转录调控信号从胞核外传至RNA滚链11(Pol II)。我们独立标记了三个亚基，建立了表达每个标记亚基的HeLa细胞系，并从核提取液中纯化了复合体。结果：1.从所有细胞系中分离纯化出一种转录超复合体，其分子量为1-2个丙二醛，并对转录激活具有积极的作用。TFIIEα中锌指的分析人类通用转录因子TFIIE由α和β亚基组成，由α-2-β-2四聚体组成，在转录起始和向伸长过渡的过程中发挥作用。TFIIEα在中央核心区有一个锌指基序，这可能对转录系统很重要。此外，在原核生物和真核生物之间的考古细菌中也证明了包含该基序的TFIIEα的N-末端区域是保守的。我们对该区域进行了核磁共振研究，发现该区域实际上形成了锌指基序，并且该区域通过其表面的负电荷在蛋白质-蛋白质相互作用中起着重要作用。POL II由起始向伸长转变的研究。由于TFIIEβC-末端螺旋区的点突变，线性化模板的转录活性已经丧失。这些突变体失去了TFIIH介导的Pol II磷酸化的刺激活性。从这些结果来看，该区域可能参与了这一转变。作用机制尚不清楚，但很可能涉及TFIIE和TFIIH之间相互作用的变化，因为TFIIH的p44与这些突变体的结合比与野生型的结合更强。

1. Characterization of the Mediator complex. The Mediator consists of more than 20 subunits and transmits the transcriptional regulation signals from outsaide of the cell nucleus to RNA rolymersae 11 (Pol II). We tagged three subunits independently, established HeLa cell lines expressing each tagged-subunit, and purified the complexes from nuclear extracts. As a result, one transcription super-complex with molecular mass 1-2 MDa was purified from all cell lines and showed positive effects on transriptionnal activation.2. Analyses of the Zn finger of TFIIEα. Human general transcription factor TFIIE consists of α and β subunits, froms an α2 β2 tetramer, and functions at transcription initiation and the transition to elongation. TFIIE α possesses a Zn finger motif at the central core region and this might be important for transcription system. Moreover, it was demonstrated that the N-terminal region of TFIIEα including this motif is conserved in Archaebacteria classifies between prokaryotes and eukaryotes. We studied this region by NMR and found that this actually forms the Zn finger motif, and that region has important roles in protein-protein interactions by negative charges on its surface.3. Studies on the transition of Pol II from initiation to elongation. By point mutations in the C-terminal helix region of TFIIEβ, the transcriptional activity with a linearized template has been lost. These mutants lost the stimulation activity of TFIIH-mediated phosphorylation of Pol II. From these results, this region might be involved in the transition. functional mechanisms are still elucive, but it is highly conceivable that the changes in interactions between TFIIE and TFIIH are involved, since the binding of p44 of TFIIH, which is essential for the transition to elongation, to those mutants became stronger than that to the wild type.