AUTOREGULATION OF SV40 EARLY GENE EXPRESSION

SV40的大T抗原与许多调节因子有关 病毒复制启动、病毒抑制等功能 转录，刺激细胞DNA和RNA合成，以及 启动和维护转型。尽管T的影响 在这些不同的代谢过程中，抗原被广泛地 有文献记载，控制这些的生化基础和分子机制 T抗原的多功能还不是很清楚。其目的是 这项研究的目的是了解T抗原是如何执行这些不同的 生化功能。我们之前描述了一系列实验 这表明T抗原在特定结合部位的相互作用 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的自动调节机制。首先，我们将通过体外作图 突变是SV40 DNA促进转录所必需的区域 体外和体内的早期和晚期基因。接下来，我们将尝试 纯化RNA聚合酶II及其选择性因子 启动子序列的特异性全酶DNA结合研究。这个 特异性提供转录选择性的机制 决定因素将在体外通过结合研究和 抄写。最后，我们将研究T抗原与 RNA聚合酶及其早期和晚期的特异性因子 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.