Graeble M A, Churcher M J, Lowe A D, Gait M J, Karn J
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6184-8. doi: 10.1073/pnas.90.13.6184.
The human immunodeficiency virus type 1 transactivator protein, tat, specifically stimulates transcription from the viral long terminal repeat. We used cell-free transcription systems to test whether tat can stimulate transcriptional read-through of an artificial terminator sequence (e.g., a stable RNA stem-loop structure followed by a tract of nine uridine residues) placed downstream of the viral long terminal repeat. In the absence of tat, RNA polymerases are prematurely released from the template at the terminator sequence. Recombinant tat protein purified from Escherichia coli increased the synthesis of full-length transcripts approximately 25-fold and decreased the amount of transcripts ending at the terminator sequence. The reaction is strictly dependent upon the presence of a functional transactivation-responsive region (TAR) sequence. Mutations in the tat binding site on TAR RNA and mutations in the TAR RNA loop block transactivation in vivo. Neither type of mutation is able to respond to tat in vitro. These results strongly suggest that after transcription through the TAR region, tat modifies the transcription complex to increase its elongation capacity.
人类免疫缺陷病毒1型反式激活蛋白tat能特异性地刺激病毒长末端重复序列的转录。我们使用无细胞转录系统来测试tat是否能刺激位于病毒长末端重复序列下游的人工终止子序列（例如，一个稳定的RNA茎环结构后接九个尿苷残基序列）的转录通读。在没有tat的情况下，RNA聚合酶在终止子序列处过早地从模板上释放。从大肠杆菌中纯化的重组tat蛋白使全长转录本的合成增加了约25倍，并减少了在终止子序列处结束的转录本数量。该反应严格依赖于功能性反式激活应答区域（TAR）序列的存在。TAR RNA上tat结合位点的突变以及TAR RNA环中的突变在体内会阻断反式激活。这两种类型的突变在体外均不能对tat作出反应。这些结果强烈表明，在转录通过TAR区域后，tat会修饰转录复合物以增加其延伸能力。
