AUTOREGULATION OF SV40 EARLY GENE EXPRESSION

SV40的大T抗原与许多调节性免疫反应有关。 功能如启动病毒复制、抑制病毒的增殖 转录，刺激细胞DNA和RNA合成，以及 转化的启动和维持。 虽然T的影响 抗原对这些不同代谢过程的影响已经被广泛地 有记录的，生物化学基础和分子机制控制这些 T抗原的多功能活性还不清楚。 目的 这项研究的目的是了解T抗原是如何进行这些不同的 生化功能。 我们之前描述了一系列实验 表明T抗原在特异性结合位点的相互作用 在抑制早期病毒mRNA的转录中起直接作用， 体外重组系统。 在这里，我们建议调查 SV40自动调节机制 首先，我们将在体外绘制 诱变SV40 DNA的区域需要促进转录 早期和晚期基因在体外和体内。 接下来，我们将尝试 纯化RNA聚合酶II及其选择性因子， 启动子序列处的特异性全酶DNA结合研究。 的 特异性提供的转录选择性机制 决定簇将在体外通过结合研究和 转录。 最后，我们将研究T抗原与 RNA聚合酶及其特异性因子在早期和晚期 启动子结合位点。

The large T antigen of SV40 has been associated with a number of regulatory functions such as initiation of viral replication, repression of viral transcription, stimulation of cellular DNA and RNA synthesis, and initiation and maintenance of transformation. Although the effects of T antigen on these diverse metabolic processes has been extensively documented, the biochemical basis and molecular mechanism governing these multifunctional activities of T antigen are not well understood. The aim of this research is to understand how T antigen carries out these diverse biochemical functions. We previously described a series of experimens which indicated that the interaction of T antigen at specific binding sites plays a direct role in repressing transcription of early viral mRNA in an in vitro reconstituted system. Here we propose to investigate the mechanism of SV40 autoregulation. First, we will map by in vitro mutagenesis the region of SV40 DNA required to promote transcription of early and late genes both in vitro and in vivo. Next, we will attempt to purify RNA polymerase II and its selectivity factors in order to perform specific holoenzyme DNA binding studies at the promoter sequences. The mechanisms of transcriptional selectivity provided by the specificity determinants will be tested in vitro both by binding studies and by transcription. Finally, we will study the interaction of T antigen with RNA polymerase and its specificity factors at the early at late promoter-binding sites.