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Analysis of gene regulatory function of TFIIF and elongin

TFIIF和elongin基因调控功能分析

基本信息

批准号：
09470519
负责人：
SHIGETAKA Kitajima
金额：
$ 8.51万
依托单位：
Tokyo Medical and Dental Univerisry
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-09470519/
关键词：
TFIIF Tripartite structure of RAP74 HIV-Tat Transcriptional elongation Transcriptional recycling Elongin Testis-specific A2 Gene targeting of elongin RNAポリメラーゼII 組織特異的因子開始型伸長型トリプシン分解組織特異的エロンガン転写制御 p32 p30 RNAポリメラーゼII

项目摘要

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) TFIIFRAP74 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) ElonginWe 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.

TFIIF可将RNA聚合酶II（Pol II）募集到基因启动子处形成的前起始复合物中，也可作为Pol II合成mRNA的延伸因子。为了了解转录延伸的调控机制，我们研究了TFIIF和elongin的功能：1）TFIIF的TFIIFRAP 74亚基具有三重结构：N端和C端结构域，以及中心区域。我们在自身免疫性疾病患者的血清中鉴定了针对TFIIF的RAP 74亚基的自身抗体，其选择性地识别RAP 74的中心部分。通过双杂交筛选，克隆了与RAP 74互作的p32基因。p32在体外能与RAP 74和HIV-Tat的中心区域结合，并能在HIV-Tat存在下促进Pol Ⅱ的延伸。p32可能与HIV的复制生长有关。通过双杂交筛选，我们未能克隆到FCP 1，它与RAP 74的C-末端结合，并使Pol II的CTD脱磷酸化。Greenblatt等报道了FCP 1。我们通过共感染RAP 30和RAP 74的杆状病毒表达并纯化了重组TFIIF。使用此，进行游离、起始和延伸形式的TFIIF的结构分析。揭示了RAP 74的三分结构、其在TFIIF中的外部定位和RAP 30的内部定位。2）elongin我们从EST数据库中克隆了一种新的elongin，A2。A2优先在睾丸中表达，而A普遍表达。重组A2能够在体外刺激Pol II的延伸。A2可能参与睾丸特异性基因的表达。此外，我们还鉴定了另一种新形式的elongin家族A3。我们现在描述它的结构和功能。我们的基因敲除项目正在研究中，我们筛选了异源的ES细胞的elongin A基因。