TRANSCRIPTION REGULATION BY SV40 SMALL T

转录因子EIIF控制一些RNA聚合酶II - 在生长细胞中诱导的转录基因，如c-myc， EGF受体和二氢叶酸还原酶。 它最初被确定为一种 结合腺病毒E2 A启动子的宿主细胞编码因子。 EIIF 可以在一个包含视网膜母细胞瘤基因产物 (Rb)和细胞周期蛋白A。Rb和细胞周期蛋白A的结合似乎抑制了 EIIF依赖性转录。 Rb和细胞周期蛋白A从EIIF中释放， 对细胞周期控制信号的反应，或在Rb或细胞周期蛋白A结合后 通过肿瘤病毒癌基因如SV 40大T抗原或腺病毒EIA。 我们以前发现SV 40小t抗原反式激活 E2 A启动子在用质粒DNA瞬时转染期间， 这需要EIIF网站。 小t不结合Rb和细胞周期蛋白A。 因此，EIIF依赖性转录的激活很可能 涉及新的未被发现的机制。 转录因子TFIIIB和TFIIIC控制一类RNA， 聚合酶III转录的基因，包括tRNA基因和 腺病毒VA-I启动子。 这些转录因子似乎也是 在生长、转化或病毒感染的细胞中活化。 小t 在瞬时转染期间反式激活VA-1启动子。 我们有 通过测试小t突变体发现，小t上的不同结构 似乎是激活E2 A和VA-I启动子所必需的。 因此，可能存在至少两个细胞过程， 小T的刺激 我们建议调查这两类的机制， 基因受到调控。 我们将使用SV 40小t作为代理进行调查 这些过程。 我们将通过凝胶位移分析来检验这些因素 使用表达小t的细胞提取物与表达小t的细胞提取物相比， 没有 在可能的情况下，我们会尝试将任何可检测到的变化 通过凝胶位移分析，体外转录增加。 我们将 继续研究小t突变体，以确定所需的结构， 转录调控 这些信息将用于确定如何 小t与细胞蛋白质相互作用，介导其效应， 转录。 我们还将检查是否有一个确定的细胞 与小t结合的蛋白质，2A型磷酸酶（PP 2A）， 通过小T介导反式激活。由于EIIF和TFIIIC因素 似乎是由磷酸化调节，这是可能的，小T 通过抑制其靶点的去磷酸化来激活转录 转录因子 这些实验将勾勒出 真核转录控制，可能导致肿瘤 其他研究尚未揭示的转变 系统.

The transcription factor, EIIF, controls some RNA polymerase II -transcribed genes that are induced in growing cells, such as c-myc, the EGF receptor, and dihydrofolate reductase. It was first identified as a host cell-encoded factor that binds to the Adenovirus E2A promoter. EIIF can be found in a complex that includes the retinoblastoma gene product (Rb) and cyclin A. Rb and cyclin A binding appear to inhibit EIIF-dependent transcription. Rb and cyclin A are released from EIIF in response to cell cycle control signals, or upon binding of Rb or cyclin A by tumor virus oncogenes such as SV40 large T antigen or Adenovirus EIA. We have previously discovered that SV40 small t antigen trans-activates the E2A promoter during transient transfection with plasmid DNA and that this requires the EIIF sites. Small t does not bind Rb and cyclin A. Therefore, activation of EIIF-dependent transcription is likely to involve novel, undiscovered mechanisms. The transcription factors, TFIIIB and TFIIIC, control a class of RNA polymerase III-transcribed genes that include tRNA genes and the Adenovirus VA-I promoter. These transcription factors also appear to be activated in growing, transformed, or virus infected cells. Small t trans-activates the VA-I promoter during transient transfection. We have found by testing small t mutants that different structures on small t appear to be required to activate the E2A and the VA-I promoters. Therefore, there may be at least two cellular processes that are stimulated by small t. We propose to investigate the mechanisms by which these two classes of genes are regulated. We will use SV40 small t as an agent to investigate these processes. We will examine these factors by gel shift analysis using extracts of cells that express small t compared to those that do not. Where possible, we will attempt to correlate any changes detectable by gel shift analysis with increased transcription in vitro. We will continue to study small t mutants to identify the structures required for transcription regulation. This information will be used to determine how small t interacts with cellular proteins the mediate its effects off transcription. We will also examine whether the one identified cellular protein that binds to small t, the enzyme type 2A phosphatase (PP2A), mediates trans-activation by small t. Since the EIIF and TFIIIC factors appear to be regulated by phosphorylation, it is possible that small t activates transcription by inhibiting the dephosphorylation of its target transcription factors. These experiments will outline pathways of eukaryotic transcriptional control that may result in neoplastic transformation which have not been revealed by investigation of other systems.