VIRAL REGULATION OF RIBOSOMAL RNA TRANSCRIPTION

描述（改编自申请人摘要）：猿猴病毒40（SV40） 大T抗原是一种多功能调节蛋白， 在病毒生命周期中的作用。 此外，大T抗原使 原代细胞，并诱导细胞转化和肿瘤形成， 动物 为了实现这些功能，大T抗原必须改变免疫原性。 控制细胞内基因表达的细胞机制 周期进展和细胞增殖。 核糖体RNA合成 聚合酶I（pol I）与细胞生长紧密相关， 以前的研究表明，大T抗原 在SV40感染的细胞中上调RNA pol I转录。 更好地 了解大T抗原如何刺激RNA聚合酶I转录，他有 建立了一个体外转录系统， 抗原的 在这些研究中，他的实验室发现， 转录因子SL 1是pol I的细胞组分之一， 大T抗原靶向的转录机器。 另外这些 研究表明，在一定程度上，UBF的磷酸化仍然是一个问题。 未鉴定的T抗原相关激酶，也可能有助于整体 刺激过程。 在这项拨款申请中，使用生物化学方法， PI建议剖析RNA聚合酶I的调节机制， 大T抗原的转录。 第一个目标是了解 大T抗原-SL1相互作用的刺激过程。 为此 他将分析缺失和单点突变的影响， 在大T抗原中存在多种体内和体外蛋白-蛋白 相互作用测定和转录测定，并将 在转录刺激下与SL1结合。 第二，他建议 使用足迹法和EMSA分析来测试这些蛋白质-蛋白质 相互作用可以增强这些细胞之间的功能性相互作用。 转录因子和促进转录的形成 起始复合体 有趣的是，他的初步研究表明， pol I刺激所必需大T抗原区域与 参与p53结合和失活的结构域。 因此， 设计了一组实验来确定潜在的功能链接 肿瘤抑制基因p53与pol I的大T抗原刺激之间的关系 转录。 最后，他将讨论的意义和作用， T抗原介导的活化过程中的UBF磷酸化， 表征相关蛋白激酶活性。 这些研究将 为哺乳动物基因的分子机制提供了新的见解 调控，并将解开一些小DNA所采取的策略， 病毒，如SV40，以改变关键细胞过程的调节。

DESCRIPTION (adapted from the applicant's abstract): Simian virus 40 (SV40) large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle. In addition, large T antigen immortalizes primary cells, and induces cell transformation and tumor formation in animals. To accomplish these functions, large T antigen has to alter the cellular mechanisms that control the expression of genes involved in cell cycle progression and cell proliferation. Ribosomal RNA synthesis by RNA polymerase I (pol I) is tightly associated with cell growth and proliferation, and previous studies demonstrated that large T antigen up-regulates RNA pol I transcription in SV40-infected cells. To better understand how large T antigen stimulates RNA pol I transcription, he has established an in vitro transcription system that responds to large T antigen. During these studies, his laboratory has discovered that the transcription factor SL1 is one of the cellular components of the pol I transcriptional machinery targeted by large T antigen. In addition, these studies suggested that, in part, the phosphorylation of UBF by a still unidentified T antigen-associated kinase, may also contribute to the overall stimulatory process. In this grant proposal, using a biochemical approach, the PI proposed to dissect the mechanism of regulation by RNA pol I transcription by large T antigen. The first goal is to understand the role of large T antigen-SL1 interaction in the stimulatory process. For this purpose, he will analyze the effects of deletion and single point mutations in large T antigen in a variety of in vivo and in vitro protein-protein interaction assays and trancription assays, and correlate the ability to bind to SL1 with the transcriptional stimulation. Second, he proposes to use footprinting and EMSA assays to test whether these protein-protein interactions may potentiate functional interactions between the transcription factors and facilitate the formation of the transcription initiation complex. Interestingly, his preliminary studies indicate that the region of large T antigen necessary for pol I stimulation overlaps with the domain involved in the binding and inactivation of p53. Thus, he has designed a set of experiments to determine the potential functional link between the tumor suppressor p53 and large T antigen stimulation of pol I transcription. Finally, he will address the significance and the role of UBF phosphorylation in the T antigen-mediated activation process and characterize the associated-protein kinase activity. These studies will provide new insights on the molecular mechanisms of mammalian gene regulation and will unravel some of the strategies adopted by small DNA viruses such as SV40 to alter the regulation of key cellular processes.