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

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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.