Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen.
Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen.
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纯化的肿瘤抗原在体外调节猿猴病毒 40 早期转录。
DOI：
10.1073/pnas.77.10.5706
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发表时间：
1980
影响因子：
11.1
通讯作者：
Tjian,R
中科院分区：
文献类型：
--
作者：
Rio,D;Robbins,A;Myers,R;Tjian,R
克隆的 DNA 模板用于通过无细胞 RNA 合成系统指导早期和晚期猿猴病毒 40 (SV40) 基因的转录。 RNA 聚合酶 II 的转录对低水平的 α-鹅膏蕈碱敏感，并且完全依赖于外源添加的 DNA 模板。当转录由含有 SV40 早期或晚期基因启动子序列的 DNA 限制性片段指导时，可以有效合成离散长度的 RNA 产物。将 D2 肿瘤抗原添加到模板 DNA 中可抑制源自 SV40 早期启动子的转录。相比之下，D2 蛋白对 SV40 或腺病毒 2 (Ad2) 晚期启动子序列的转录影响很小或没有影响。当含有SV40早期启动子和Ad2晚期启动子的克隆DNA混合物用于指导RNA合成时，D2蛋白特异性抑制SV40早期基因的合成，但不抑制Ad2晚期序列的合成。 D2 DNA 结合蛋白对 SV40 突变模板指导的转录也没有影响，该模板包含完整的早期启动子序列，但缺乏特定的肿瘤抗原结合位点。我们已经证实，在转录测定条件下，D2 蛋白与其在野生型模板 DNA 上的识别位点特异性结合并相互作用，但无法与缺乏含有 SV40 肿瘤抗原结合位点的序列的突变体或 Ad2 DNA 模板结合。这些发现提供了证据，证明肿瘤抗原与其在 DNA 上的特异性结合位点之间的直接相互作用是 SV40 A 基因自动调节其转录的机制。
Cloned DNA templates were used to direct the transcription of early and late simian virus 40 (SV40) genes by a cell-free RNA-synthesizing system. Transcription by RNA polymerase II was sensitive to low levels of alpha-amanitin and completely dependent on exogenously added DNA template. RNA products of discrete lengths were efficiently synthesized when transcription was directed by DNA restriction fragments containing promoter sequences for either early or late genes of SV40. Addition of the D2 tumor antigen to the template DNA inhibited transcription originating from the SV40 early promoter. In contrast, the D2 protein had little or no effect on the transcription from SV40 or adenovirus 2 (Ad2) late promoter sequences. When a mixture of cloned DNA containing SV40 early promoter and Ad2 late promoter was used to direct RNA synthesis, the D2 protein specifically inhibited the synthesis of SV40 early genes but not that of Ad2 late sequences. The D2 DNA binding protein also had no effect on the transcription directed by SV40 mutant templates that contain an intact early promoter sequence but lack specific tumor-antigen binding sites. We have confirmed that, under the conditions of the transcription assay, the D2 protein binds and interacts specifically with its recognition sites on wild-type template DNAs but fails to bind to mutant or Ad2 DNA templates that lack sequences containing SV40 tumor-antigen binding sites. These findings provide evidence that a direct interaction between tumor antigen and its specific binding sites on DNA is the mechanism by which the SV40 A gene autoregulates its transcription.