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

各种转录超复合体基因表达组织规律机制的生化和遗传学分析。

基本信息

批准号：
13470489
负责人：
OHKUMA Yoshiaki
金额：
$ 9.22万
依托单位：
Osaka university
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

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.

1. 中介复合物的表征。中介体由20多个亚基组成，将细胞核外的转录调控信号传递给RNA聚合酶11 （Pol II）。我们独立标记了三个亚基，建立了表达每个亚基的HeLa细胞系，并从核提取物中纯化了复合物。结果，从所有细胞系中纯化出一个分子量为1-2 MDa的转录超复合体，并显示出转录激活的积极作用。TFIIEα锌指分析。人通用转录因子TFIIE由α2 β2四聚体的α和β亚基组成，在转录起始和转录延伸过渡中起作用。TFIIE α在中心核心区有一个Zn指基序，这可能对转录系统很重要。此外，研究表明，包含该基序的TFIIEα的n端区域在古细菌中是保守的，分为原核生物和真核生物。我们通过核磁共振研究了这一区域，发现这实际上形成了Zn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重要作用。Pol II从起始到延伸转变的研究。通过在TFIIEβ的c端螺旋区发生点突变，失去了线性化模板的转录活性。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，这个区域可能参与了转变。功能机制尚不清楚，但很有可能与TFIIE和TFIIH之间相互作用的变化有关，因为TFIIH的p44结合对这些突变体的结合比对野生型的结合更强，而p44是向伸长过渡所必需的。