ACS Chem Biol. 2019 Oct 18;14(10):2264-2275. doi: 10.1021/acschembio.9b00610. Epub 2019 Oct 7.
The C-terminal domain (CTD) of the largest subunit in eukaryotic RNA polymerase II has a repetitive heptad sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 which is responsible for recruiting transcriptional regulatory factors. The seventh heptad residues in mammals are less conserved and subject to various post-translational modifications, but the consequences of such variations are not well understood. In this study, we use ultraviolet photodissociation mass spectrometry, kinetic assays, and structural analyses to dissect how different residues or modifications at the seventh heptad position alter Tyr1 phosphorylation. We found that negatively charged residues in this position promote phosphorylation of adjacent Tyr1 sites, whereas positively charged residues discriminate against it. Modifications that alter the charges on seventh heptad residues such as arginine citrullination negate such distinctions. Such specificity can be explained by conserved, positively charged pockets near the active sites of ABL1 and its homologues. Our results reveal a novel mechanism for variations or modifications in the seventh heptad position directing subsequent phosphorylation of other CTD sites, which can contribute to the formation of various modification combinations that likely impact transcriptional regulation.
真核生物 RNA 聚合酶 II 大亚基的 C 末端结构域（CTD）具有 Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 的重复七肽序列，该序列负责招募转录调控因子。哺乳动物中第七个七肽残基的保守性较低，并且会受到各种翻译后修饰的影响，但这些变化的后果尚不清楚。在这项研究中，我们使用紫外光解离质谱、动力学测定和结构分析来剖析第七个七肽位置的不同残基或修饰如何改变 Tyr1 的磷酸化。我们发现该位置的带负电荷的残基促进相邻 Tyr1 位点的磷酸化，而带正电荷的残基则抑制磷酸化。改变第七个七肽残基电荷的修饰，如精氨酸瓜氨酸化，会否定这种区分。这种特异性可以用 ABL1 及其同源物的活性位点附近保守的带正电荷的口袋来解释。我们的结果揭示了一种新的机制，即第七个七肽位置的变化或修饰指导其他 CTD 位点的后续磷酸化，这可能有助于形成各种修饰组合，从而可能影响转录调控。
