The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.
The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.
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DOI：
10.1073/pnas.93.1.326
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发表时间：
1996-01
影响因子：
11.1
通讯作者：
J. Zou;P. Luciw
中科院分区：
文献类型：
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作者：
J. Zou;P. Luciw
猴泡沫病毒1型的转录反式激活因子（Tas）强烈增强由病毒长末端重复序列中的启动子和位于env基因内的新发现的内部启动子指导的基因表达。位于内部启动子中TATA盒5'端的121 bp区域是Tas反式激活所必需的。本研究的目的是确定该区域中精确的Tas响应靶标，并确定Tas在转录调控中的作用。通过对小鼠和猿猴细胞中瞬时表达试验中的簇集位点突变和杂交启动子的分析，显示这121 bp区域内的两个独立的序列元件是Tas-dependent转录增强子。这些靶，每个< 30 bp的长度和显示没有明显的序列同源性，彼此，被指定为启动子近端和启动子远端元件。通过凝胶电泳迁移率变动分析，使用纯化的谷胱甘肽S-转移酶-Tas融合蛋白在大肠杆菌中表达，目标近端的TATA盒表现出强结合谷胱甘肽S-转移酶-Tas，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被来自DNase I的谷胱甘肽S-转移酶-Tas保护。我们提出了一个模型，用于猴泡沫病毒1型内部启动子的反式激活，其中Tas直接与位于TATA盒5'端的近端靶元件相互作用。在该模型中，假定与该元件连接的Tas与细胞RNA聚合酶II起始复合物的组分相互作用，从而增强由病毒内部启动子指导的转录。
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.