Majello B, Napolitano G, Giordano A, Lania L
Department of Genetics, Molecular and General Biology, University of Naples 'Federico II' and International Institute of Genetics and Biophysics, CNR, Naples, Italy.
Oncogene. 1999 Aug 12;18(32):4598-605. doi: 10.1038/sj.onc.1202822.
The CDK9 kinase in association with Cyclin T is a component of the transcription positive-acting complex pTEFb which facilitates the transition from abortive to productive transcription elongation by phosphorylating the carboxyl-terminal domain of RNA polymerase II. The Cyclin T1/CDK9 complex is implicated in Tat transactivation, and it has been suggested that Tat functions by recruiting this complex to RNAPII through cooperative binding to RNA. Here, we demonstrate that targeted recruitment of Cyclin T1/CDK9 kinase complex to specific promoters, through fusion to a DNA-binding domain of either Cyclin T1 or CDK9 kinase, stimulates transcription in vivo. Transcriptional enhancement was dependent on active CDK9, as a catalytically inactive form had no transcriptional effect. We determined that, unlike conventional activators, DNA-bound CDK9 does not activate enhancerless TATA-promoters unless TBP is overexpressed, suggesting that CDK9 acts in vivo at a step subsequent to TFIID recruitment DNA-bound. Finally, we determined that CDK9-mediated transcriptional activation is mediated by preferentially stimulating productive transcription elongation.
与细胞周期蛋白T相关的CDK9激酶是转录正性作用复合物pTEFb的一个组成部分，该复合物通过磷酸化RNA聚合酶II的羧基末端结构域促进从流产型转录延伸到有效转录延伸的转变。细胞周期蛋白T1/CDK9复合物与Tat反式激活有关，并且有人提出Tat通过与RNA协同结合将该复合物招募到RNA聚合酶II发挥作用。在这里，我们证明，通过与细胞周期蛋白T1或CDK9激酶的DNA结合结构域融合，将细胞周期蛋白T1/CDK9激酶复合物靶向招募到特定启动子，可在体内刺激转录。转录增强依赖于活性CDK9，因为催化失活形式没有转录作用。我们确定，与传统激活剂不同，结合DNA的CDK9不会激活无增强子的TATA启动子，除非TBP过表达，这表明CDK9在体内TFIID招募到结合DNA之后的步骤发挥作用。最后，我们确定CDK9介导的转录激活是通过优先刺激有效转录延伸来介导的。
