Structural Motifs for CTD Kinase Specificity on RNA Polymerase II during Eukaryotic Transcription.

The phosphorylation states of RNA polymerase II coordinate the process of eukaryotic transcription by recruitment of transcription regulators. The individual residues of the repetitive heptad of the C-terminal domain (CTD) of the biggest subunit of RNA polymerase II are phosphorylated temporally at different stages of transcription. Intriguingly, despite similar flanking residues, phosphorylation of Ser2 and Ser5 in CTD heptads play dramatically different roles. The mechanism of how the kinases place phosphorylation on the correct serine is not well understood. In this paper, we use biochemical assays, mass spectrometry, molecular modeling, and structural analysis to understand the structural elements determining which serine of the CTD heptad is subject to phosphorylation. We identified three motifs in the activation/P+1 loops differentiating the intrinsic specificity of CTD in various CTD kinases. We characterized the enzyme specificity of the CTD kinases—CDK7 as Ser5-specific, Erk2 with dual specificity for Ser2 and Ser5, and Dyrk1a as a Ser2-specific kinase. We also show that the specificities of kinases are malleable and can be modified by incorporating mutations in their activation/P+1 loops that alter the interactions of the three motifs. Our results provide an important clue to the understanding of post-translational modification of RNA polymerase II temporally during active transcription.

RNA聚合酶II的磷酸化状态通过招募转录调节因子来协调真核生物转录过程。RNA聚合酶II最大亚基的C末端结构域（CTD）重复七肽中的单个残基在转录的不同阶段会适时地被磷酸化。有趣的是，尽管侧翼残基相似，但CTD七肽中Ser2和Ser5的磷酸化却起着截然不同的作用。激酶如何将磷酸化作用于正确的丝氨酸的机制尚不清楚。在本文中，我们利用生化分析、质谱分析、分子建模和结构分析来了解决定CTD七肽中哪个丝氨酸会被磷酸化的结构要素。我们在激活/P + 1环中鉴定出三个基序，它们区分了各种CTD激酶中CTD的内在特异性。我们对CTD激酶的酶特异性进行了表征——CDK7为Ser5特异性，Erk2对Ser2和Ser5具有双重特异性，Dyrk1a为Ser2特异性激酶。我们还表明，激酶的特异性是可塑的，可以通过在其激活/P + 1环中引入改变三个基序相互作用的突变来进行修饰。我们的研究结果为理解在活跃转录过程中RNA聚合酶II的翻译后修饰的时间顺序提供了重要线索。