Direct evidence that MIV-1 tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation
Direct evidence that MIV-1 tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation
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DOI：
10.1006/jmbi.1999.2933
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发表时间：
1999-07-30
影响因子：
5.6
通讯作者：
Karn, J
中科院分区：
文献类型：
--
作者：
Isel, C;Karn, J
人类免疫缺陷？1型病毒（HIV-1） Tat蛋白通过刺激RNA聚合酶活性调控转录。使用固定模板，我们已经能够研究Tat在HIV-1转录起始前和延伸阶段对蛋白激酶活性的影响。在HIV-1 LTR形成的起始前复合物中，RNA聚合酶II的c端结构域（CTD）被转录因子IIH （TFIIH）快速磷酸化。在起始前复合物中，Tat的加入既不影响CTD磷酸化的速率也不影响其磷酸化程度。相比之下，Tat能够在延伸复合物中刺激额外的CTD磷酸化。这个反应产生了一种新的RNA聚合酶，我们称之为RNA聚合酶IIo*。RNA聚合酶IIo*的形成仅发生在携带功能性TAR RNA元件的模板转录后，并且被低浓度的5,6-二氯-1- β -d -核糖呋喃基苯并咪唑（DRB）强烈抑制，DRB是一种有效的CDK9抑制剂，是tat相关激酶（TAK）的蛋白激酶亚基。免疫印迹实验表明，CDK9及其相关的细胞周期蛋白，细胞周期蛋白T-1，在起始前复合物和延伸复合物中都以相同的水平存在。我们得出结论，CDK9激酶的激活，导致CTD磷酸化，只发生在通过tat识别元件TAR RNA转录的延伸复合物中。(C) 1999学术出版社。
The human immunodeficiency? virus type-1 (HIV-1) Tat protein regulates transcription by stimulating RNA polymerase processivity. Using immobilised templates, we have been able to study the effects of Tat on protein kinase activity during the pre-initiation and elongation stages of HIV-1 transcription. In pre-initiation complexes formed at the HIV-1 LTR, the C-terminal domain (CTD) of RNA polymerase II is rapidly phosphorylated by transcription factor IIH (TFIIH). Addition of Tat does not affect either the rate or the extent of CTD phosphorylation in the preinitiation complexes. By contrast, Tat is able to stimulate additional CTD phosphorylation in elongation complexes. This reaction creates a novel form of the RNA polymerase that we have called RNA polymerase IIo*. Formation of the RNA polymerase IIo* occurs only after transcription of templates carrying a functional TAR RNA element and is strongly inhibited by low concentrations of 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a potent inhibitor of CDK9, the protein kinase subunit of the Tat-associated kinase (TAK). Immunoblotting experiments have shown that CDK9 and its associated cyclin, cyclin T-1, are present at equivalent levels in both the pre-initiation and elongation complexes. We conclude that activation of the CDK9 kinase, leading to CTD phosphorylation, occurs only in elongation complexes that have transcribed through the Tat-recognition element, TAR RNA. (C) 1999 Academic Press.