MECHANISM OF P53 SILENCING BY ADENOVIRUS E1B 55K PROTEIN

P53是一种序列特异性的DNA结合转录因子，它 DNA对细胞周期的抑制和诱导细胞凋亡的作用 损坏。这两个p53等位基因在转移性人类中都经常发生突变。 癌症表明P53是一种肿瘤抑制因子。这很可能是 因为P53的失活允许突变和基因的积累 激活癌基因导致肿瘤进展的扩增。 腺病毒E1B 55K蛋白与P53结合并抑制P53反式激活 通过特定的蛋白质-蛋白质相互作用， 对55K的致癌转化活性至关重要 腺病毒E1a。我们建议研究两种p53基因的分子机制。 体外研究激活和E1B 55K转录沉默 与纯化的通用转录因子结合。与之结合的多肽 P53的相互作用及其刺激机制 笔录将会确定。塔塔-盒子装订的表面 P53结合的蛋白质(Tcp)将通过分析P53来确定 结合到具有特定改变的大量突变Tbps集合 表面残留物。将分析与纯化的TFIID-TAFs的相互作用 通过蛋白质印迹和化学交联法。功能目标将是 通过确定哪些纯化因子可以克服 高浓度P53对转录的抑制作用。琼脂糖凝胶 大型DNA-蛋白质复合体的电泳和足迹将被 用来分析P53对起始复合体组装和转录的影响 引发后引发复合体的解离。自.以来 最近的研究表明，55K含有一个抑制结构域，即LexA-55K 融合蛋白将得到表达和纯化，其抑制能力 将对抄写进行调查。如果基础转录被抑制 55K，将确定被55K抑制的通用转录因子 并定义了特定的交互。如果激活的转录是 明确抑制、55K抑制的辅活化子将被识别 并对其抑制机理进行了研究。了解其作用机制 P53的转录激活和腺病毒E1B 55K的抑制作用 可能允许设计重新激活人类肿瘤中的p53的治疗方法， 导致细胞周期停滞和肿瘤细胞的凋亡。

P 53 is a sequence-specific DNA-binding transcription factor which functions to arrest the cell cycle and induce apoptosis in response to DNA damage. Both p 53 alleles are frequently mutated in metastatic human cancers indicating that p53 is a tumor suppressor. This is probably because inactivation of p53 allows the accumulation of mutations and gene amplifications which activate oncogenes resulting in tumor progression. Adenovirus E1B 55K protein binds to p53 and represses p53 transactivation through specific protein-protein interactions, activities which are critical for the oncogenic transforming activity of 55K in conjunction with adenovirus E1A. We propose to study the molecular mechanisms of both p 53 activation and E1B 55K transcriptional silencing through in vitro studies with purified general transcription factors. The polypeptides with which p53 interacts and the mechanism by which these interactions stimulate transcription will be determined. The surface of the TATA-box binding protein (TCP) to which p 53 binds will be determined by analyzing p53 binding to a large collection of mutant TBPs with alterations in specific surface residues. Interactions with purified TFIID-TAFs will be analyzed by protein blotting and chemical cross-linking. Functional targets will be identified by determining which purified factors can overcome transcriptional squelching by high concentrations of p53. Agarose gel electrophoresis of large DNA-protein complexes and footprinting will be used to analyze the influence of p53 on initiation complex assembly and on disassociation of the initiation complex following initiation. Since recent work has shown that 55K contains a repression domain, a LexA-55K fusion protein will be expressed and purified, and its ability to repress transcription will be investigated. If basal transcription in inhibited by 55K, the general transcription factors inhibited by 55K will be identified and specific interactions defined. If activated transcription is specifically inhibited, coactivators inhibited by 55K will be identified and the mechanisms of inhibition studied. Understanding the mechanism of transcriptional activation by p53 and its inhibition by adenovirus E1B 55K may allow the design of therapies which reactivate p53 in human tumors, leading to cell-cycle arrest and apoptosis of tumor cells.