Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type I Tat-activated transcriptional elongation
Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type I Tat-activated transcriptional elongation
复制标题
DOI：
10.1128/mcb.22.13.4622-4637.2002
复制
发表时间：
2002-07-01
影响因子：
5.3
通讯作者：
Karn, J
中科院分区：
文献类型：
--
作者：
Kim, YK;Bourgeois, CF;Karn, J
人类免疫缺陷病毒I型Tat蛋白刺激转录延伸是由CDK9介导的，CDK9是一种磷酸化RNA聚合酶II羧基末端结构域（CTD）的激酶。为了获得这种磷酸化事件可以改变RNA聚合酶的直接证据，我们制备了被lac抑制因子阻止的转录延伸复合物。然后用蛋白磷酸酶1处理CTD去磷酸化。当IPTG（异丙基- β - d -硫代半乳糖苷）和核苷酸加入到反应中时，去磷酸化的转录复合物能够恢复转录延伸。在这些追逐条件下，在含有Tat蛋白的复合物中观察到CTD的有效再磷酸化，而在没有Tat蛋白的情况下制备的转录复合物中则没有。合成肽的免疫印迹和激酶分析表明，Tat直接激活了CDK9，因为在Tat存在或不存在的情况下制备的转录复合物中，该酶及其细胞周期蛋白伴侣细胞周期蛋白T1的水平相当。在CDK9激酶抑制剂DRB（5,6-二氯-1- β -d -核糖呋喃基-苯并咪唑）存在的情况下，对去磷酸化的延伸转录复合物进行Chase实验。在这些条件下，CTD在伸长过程中没有再磷酸化，并且通过茎环终止器或弯曲DNA捕获序列的转录被强烈抑制。在实验中，CTD在延伸之前被磷酸化，终端序列的读通量与CTD修饰的程度成正比。加工能力的变化仅仅是由于CTD磷酸化，因为即使在去除CDK9的第二底物Spt5之后，RNA聚合酶的延伸也会被tat激活的CDK9活性增强。我们得出结论，CDK9对RNA聚合酶11ctd的磷酸化直接增强了转录延伸。
Stimulation of transcriptional elongation by the human immunodeficiency virus type I Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-beta-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase 11 CTD by CDK9 enhances transcription elongation directly.