Zou J X, Luciw P A
Department of Medical Pathology, University of California, Davis 95616, USA.
Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):326-30. doi: 10.1073/pnas.93.1.326.
The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene. A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas. The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation. By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers. These targets, each < 30 bp in length and displaying no apparent sequence homology one to the other, are designated the promoter-proximal and promoter-distal elements. By means of the gel electrophoresis mobility-shift assays, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind. In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I. We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box. In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter.
1型猿猴泡沫病毒的转录反式激活因子（Tas）能强力增强由病毒长末端重复序列中的启动子以及env基因内新发现的内部启动子所指导的基因表达。内部启动子中TATA框紧邻的5'端有一个121 bp的区域，是Tas反式激活所必需的。本研究旨在确定该区域中精确的Tas反应靶点，并确定Tas在转录调控中的作用。通过在鼠类和猿猴细胞的瞬时表达试验中分析成簇位点突变和杂交启动子，结果表明该121 bp区域内的两个独立序列元件是依赖Tas的转录增强子。这些靶点，每个长度<30 bp，彼此之间没有明显的序列同源性，分别被命名为启动子近端元件和启动子远端元件。通过凝胶电泳迁移率变动分析，利用在大肠杆菌中表达的纯化谷胱甘肽S-转移酶-Tas融合蛋白，TATA框近端的靶点与谷胱甘肽S-转移酶-Tas表现出强结合，而远端元件似乎不结合。此外，足迹分析显示启动子近端元件中的26 bp被谷胱甘肽S-转移酶-Tas保护不被DNase I切割。我们提出了一个1型猿猴泡沫病毒内部启动子反式激活的模型，其中Tas直接与紧邻TATA框5'端的近端靶点元件相互作用。在这个模型中，附着在该元件上的Tas被推测与细胞RNA聚合酶II起始复合物的一个或多个组分相互作用，从而增强由病毒内部启动子指导的转录。
