Analysis of gene regulatory function of TFIIF and elongin

TFIIF将RNA聚合酶II(Pol II)招募到基因启动子形成的起始前复合体中，也可以作为Pol II合成mRNA的延伸因子，Elongin是一种通用的延伸因子，可能参与了VHL疾病的肿瘤发生。为了了解转录调控机制，本研究对TFIIF和细长蛋白的功能进行了研究。1)TFIIF的TFIIFRAP74亚基被预测为三段结构，N-末端和C-末端结构域，以及中心区。我们从自身免疫性疾病患者的血清中鉴定出针对TFIIF的RAP74亚单位的自身抗体，这些抗体选择性地识别RAP74的中心部分。经双杂交筛选，p32被克隆为RAP74相互作用基因。在体外，P32能与RAP74和HIV-TAT的中心区结合，并能在HIV-TAT存在的情况下加速Pol II的延伸。P32可能与HIV的复制生长有关。通过双杂交筛选，我们无法克隆与RAP74的C末端结合并使POL II的CTD去磷酸化的FCP1。FCP1由Greenblatt等人报道。我们通过杆状病毒共感染RAP30和RAP74表达并纯化了重组TFIIF。在此基础上，对TFIIF的自由态、起始态和延伸态进行了结构分析。揭示了RAP74的三聚体结构及其在TFIIF中的外部定位和RAP30的内部定位。我们正在尝试结晶TFIIF分子用于结构分析2)Elongin我们从数据库中的EST克隆了一种新形式的细长蛋白A2。A2在睾丸中优先表达，而A在睾丸中普遍表达。重组A2能在体外刺激Pol II的伸长。A2可能在睾丸特异基因的表达中起作用。此外，我们还发现了细长蛋白家族的另一种新形式--A3。我们现在正在描述它的结构和功能。我们的基因敲除计划正在研究中，我们筛选出了细长蛋白A基因的异源ES细胞。

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.