Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription
Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription
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DOI：
10.1074/jbc.ra119.007697
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发表时间：
2019-05-24
影响因子：
4.8
通讯作者：
Zhang, Yan
中科院分区：
文献类型：
--
作者：
Irani, Seema;Sipe, Sarah N.;Zhang, Yan
RNA聚合酶II的c端结构域（CTD）包含一个重复七肽序列（YSPTSPS），其磷酸化状态通过招募蛋白质调节因子来协调真核生物的转录。在CTD的特定残基上精确地放置和去除磷酸基团对于RNA聚合酶ii介导的转录的保真度和有效性至关重要。在转录延伸过程中，磷酸化丝氨酸(5)（pSer(5)）被CTD磷酸酶逐渐去磷酸化，而丝氨酸(2)磷酸化则逐渐积累。利用质谱、x射线晶体学、蛋白质工程和免疫印迹分析，我们研究了SSU72同源物RNA聚合酶II CTD磷酸酶（SSU72，来自果蝇黑ogaster）的结构和功能，这是一种重要的CTD磷酸酶，在从延伸到终止的过渡过程中去磷酸化pSer(5)，以确定SSU72区分高度相似的pSer(2)和pSer(5) CTD的机制。我们发现Ssu72可以有效地去磷酸化pSer(5)，但对pSer(7)和pSer(2)的活性较低。结构分析表明，Ssu72要求底物SP基序上的脯氨酸残基呈顺式构型，形成一个紧旋，才能被Ssu72识别。我们还注意到SP基序两侧的残基，如靠近Ser(2)的庞大的Tyr(1)，阻止了这种构型的形成，并使Ssu72能够区分不同的SP基序。Tyr(1)的磷酸化进一步阻止了Ssu72与pSer(2)的结合，从而阻止了Ser(2)过早的去磷酸化。我们的研究结果揭示了Tyr(1)在区分RNA聚合酶II中CTD的Ser(2)/Ser(5)的磷酸化状态方面的关键作用，这些磷酸化状态发生在转录的不同阶段。
The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser(5) (pSer(5)) is gradually dephosphorylated by CTD phosphatases, whereas Ser(2) phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer(5) at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer(2) and pSer(5) CTDs. We found that Ssu72 dephosphorylates pSer(5) effectively but only has low activities toward pSer(7) and pSer(2). The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight -turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr(1) next to Ser(2), prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr(1) further prohibited Ssu72 binding to pSer(2) and thereby prevented untimely Ser(2) dephosphorylation. Our results reveal critical roles for Tyr(1) in differentiating the phosphorylation states of Ser(2)/Ser(5) of CTD in RNA polymerase II that occur at different stages of transcription.