| Co-Investigator(Kenkyū-buntansha) |
ATORU Oshiro Medical Research Institue, Tokyo Medical and Dental Univerisry Assistant Professor, 難治疾患研究所, 助手 (30160485)
TERUMASA Tuchiya Medical Research Insitute, Tokyo Medical and Dental Univerisry Assistant Professor, 難治疾患研究所, 助手 (20242109)
YUKIO Yasukochi Medical Research Institue, Tokyo Medical and Dental Univerisry Professor, 難治疾患研究所, 教授 (60037398)
TEIJIRO Aso Cancer Research Institue, Investigator, 癌研究会研究所, 研究員 (20291289)
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| Research Abstract |
TFIIF, which recruits RNA polymerase II (Pol II) into preinitiation complex formed at gene promoter, can also function as an elongation factor for mRNA synthesis by Pol II, Elongin is a general elongation factor which may be involved in oncogenesis in VHL disease. To understand regu1atory mechanism of transcriptional elonagtion, we investigated function of TFIIF and elongin in this study.
1) TFIIF
RAP74 subunit of TFIIF is predicted to have tri-partite structure ; N- and C-terminal domains, and central region. We identified autoantibodies against RAP74 subunit of TFIIF in sera from autoimmune disease patients which selectively recognize the central portion of RAP74. By 2-hybrid screen, p32 was cloned as RAP74-interacting gene. p32 could bind to central region of RAP74 and HIV-Tat in vitro, and was capable for accelerating elongation by Pol II in the presence of HIV-Tat. p32 may be implicated in replicative growth of HIV.We could not clone FCP1, which binds to C-terminal of RAP74 and dephosphorylates CTD of Pol II by 2-hybrid screening. FCP1 was reported by Greenblatt et al. We expressed and purified recombinant TFIIF through co-infection of baculoviruses for RAP30 and 74. Using this, structural analysis of free, initiation, and elongation forms of TFIIF was performed. Tripartite structure of RAP74, its outer localization in TFIIF, and inner location of RAP30 was revealed. We are trying to crystallize TFIIF molecule for structural analysis.
2) Elongin
We cloned a novel form of elongin, A2, from EST on database. A2 is expressed preferentially in the testis while A is ubiquitously expressed. Recombinant A2 is capable for stimulating elongation of Pol II in vitro. A2 may function in expression of testis-specific genes. Furthermore, we identified A3, another novel form of elongin family. We are now characterizing its strucutre and function. Our gene knock-out project is under investigation, We screened ES cells which are heterologous for elongin A gene.
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