Napolitano G, Majello B, Licciardo P, Giordano A, Lania L
Department of Genetics, Molecular and General Biology, University of Naples 'Federico II' and International Institute of Genetics and Biophysics, CNR, Via Mezzocannone 8, 80134, Naples, Italy.
Gene. 2000 Aug 22;254(1-2):139-45. doi: 10.1016/s0378-1119(00)00278-x.
Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) is an important step in transcription and the positive transcription elongation factor b (P-TEFb) has been proposed to facilitate elongation at many genes. The P-TEFb contains a catalytic subunit (Cdk9) that, in association with a cyclin subunit (cyclinT1), has the ability to phosphorylate the CTD substrate in vitro. Here, we demonstrate that cyclinT1/Cdk9-mediated transcription requires CTD-containing RNAPII, suggesting that the CTD is the major target of the cyclinT1/Cdk9 complex in vivo. Unlike Cdk7 and Cdk8, two other cyclin-dependent kinases that are capable of phosphorylating the CTD in vitro, we found that only the Cdk9 activates gene expression in a catalysis-dependent manner. Finally, unlike cyclinT1 and T2, we found that the targeted recruitment to promoter DNA of cyclinK (a recently described alternative partner of Cdk9) does not stimulate transcription in vivo. Collectively, our data strongly indicate that the P-TEFb kinase subunits cyclinT/Cdk9 are specifically involved in transcription and the CTD domain of RNAPII is the major functional target of this complex in vivo.
RNA聚合酶II（RNAPII）羧基末端结构域（CTD）的磷酸化是转录过程中的重要步骤，并且有研究提出正性转录延伸因子b（P-TEFb）有助于许多基因的转录延伸。P-TEFb包含一个催化亚基（Cdk9），其与一个细胞周期蛋白亚基（细胞周期蛋白T1）结合，在体外具有磷酸化CTD底物的能力。在此，我们证明细胞周期蛋白T1/Cdk9介导的转录需要含有CTD的RNAPII，这表明CTD是细胞周期蛋白T1/Cdk9复合物在体内的主要作用靶点。与另外两种能够在体外磷酸化CTD的细胞周期蛋白依赖性激酶Cdk7和Cdk8不同，我们发现只有Cdk9以催化依赖的方式激活基因表达。最后，与细胞周期蛋白T1和T2不同，我们发现细胞周期蛋白K（最近描述的Cdk9的替代伴侣）靶向募集到启动子DNA在体内不会刺激转录。总体而言，我们的数据强烈表明P-TEFb激酶亚基细胞周期蛋白T/Cdk9特异性参与转录，并且RNAPII的CTD结构域是该复合物在体内的主要功能靶点。
