MECHANISM OF P53 SILENCING BY ADENOVIRUS E1B 55K PROTEIN

p53是一种序列特异性dna结合转录因子

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.