Keen N J, Churcher M J, Karn J
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
EMBO J. 1997 Sep 1;16(17):5260-72. doi: 10.1093/emboj/16.17.5260.
The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.
HIV-1反式激活蛋白Tat是转录延伸的强效激活剂。在其穿过反式激活应答区域（TAR）的过程中，Tat因其能够直接结合新生RNA链上表达的TAR RNA而被招募到延伸的RNA聚合酶上。我们已经表明，获得Tat的转录复合物产生的全长转录本比未接触Tat的复合物多3倍。蛋白质免疫印迹实验表明，Tat与暂停的聚合酶紧密相关。为了确定TAR RNA是否也附着在转录复合物上，将DNA寡核苷酸与停滞复合物上的新生链退火，然后用RNase H切割RNA。切割后，携带TAR RNA的新生链的5'端被定量去除，但转录本的3'端仍与转录复合物相关。即使去除TAR RNA后，被Tat激活的转录复合物仍显示出增强的持续合成能力。我们得出结论，Tat与细胞辅助因子一起附着在转录复合物上并刺激持续合成能力，而TAR RNA在延伸激活中不发挥直接作用，仅用于招募Tat和细胞辅助因子。
