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Regulation of the DEAH/RHA helicase Prp43 by the G-patch factor Pfa1
Proceedings of the National Academy of Sciences of the United States of America ( IF 11.1 ) Pub Date : 2022-11-21 , DOI: 10.1073/pnas.2203567119
Marieke Enders, Ralf Ficner, Sarah Adio

The DEAH/RHA helicase Prp43 remodels protein–RNA complexes during pre-messenger RNA (mRNA) splicing and ribosome biogenesis. The helicase activity and ATP turnover are intrinsically low and become activated by G-patch (gp) factors in the specific cellular context. The gp motif connects the helicase core to the flexible C-terminal domains, but it is unclear how this affects RecA domain movement during catalysis and the unwinding of RNA substrates. We developed single-molecule Förster Resonance Energy Transfer (smFRET) reporters to study RecA domain movements within Prp43 in real time. Without Pfa1(gp), the domains approach each other adopting predominantly a closed conformation. The addition of Pfa1(gp) induces an open state, which becomes even more prevalent during interaction with RNA. In the open state, Prp43 has reduced contacts with bound nucleotide and shows rapid adenosine diphosphate (ADP) release accelerating the transition from the weak (ADP) to the strong (apo) RNA binding state. Using smFRET labels on the RNA to probe substrate binding and unwinding, we demonstrate that Pfa1(gp) enables Prp43(ADP) to switch between RNA-bound and RNA-unbound states instead of dissociating from the RNA. ATP binding to the apo-enzyme induces the translocation along the RNA, generating the unwinding force required to melt proximal RNA structures. During ATP turnover, Pfa1(gp) stimulates alternating of the RecA domains between open and closed states. Consequently, the translocation becomes faster than dissociation from the substrate in the ADP state, allowing processive movement along the RNA. We provide a mechanistic model of DEAH/RHA helicase motility and reveal the principles of Prp43 regulation by G-patch proteins.

中文翻译:

G 贴片因子 Pfa1 对 DEAH/RHA 解旋酶 Prp43 的调控

DEAH/RHA 解旋酶 Prp43 在前信使 RNA (mRNA) 剪接和核糖体生物合成过程中重塑蛋白质-RNA 复合物。解旋酶活性和 ATP 转换本质上很低,并在特定细胞环境中被 G 补丁 (gp) 因子激活。gp 基序将解旋酶核心连接到灵活的 C 末端结构域,但尚不清楚这如何影响催化过程中的 RecA 结构域运动和 RNA 底物的解旋。我们开发了单分子 Förster 共振能量转移 (smFRET) 报告基因来实时研究 Prp43 中的 RecA 域运动。没有 Pfa1(gp),域相互接近,主要采用封闭构象。添加 Pfa1(gp) 会导致开放状态,这在与 RNA 相互作用期间变得更加普遍。在打开状态下,Prp43 减少了与结合核苷酸的接触,并显示二磷酸腺苷 (ADP) 的快速释放加速了从弱 (ADP) 到强 (apo) RNA 结合状态的转变。使用 RNA 上的 smFRET 标签来探测底物结合和解旋,我们证明 Pfa1(gp) 使 Prp43(ADP) 能够在 RNA 结合和 RNA 未结合状态之间切换,而不是从 RNA 解离。ATP 与脱辅基酶的结合会诱导 RNA 的易位,从而产生解链近端 RNA 结构所需的解旋力。在 ATP 周转期间,Pfa1(gp) 刺激 RecA 域在打开和关闭状态之间交替。因此,易位变得比从 ADP 状态的底物解离更快,从而允许沿着 RNA 进行连续运动。
更新日期:2022-11-21
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