Department of Molecular Structural Biology, Institute of Microbiology and Genetics, Georg-August-University Göttingen, D-37077 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2022 Nov 29;119(48):e2203567119. doi: 10.1073/pnas.2203567119. Epub 2022 Nov 21.
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.
DEAH/RHA 解旋酶 Prp43 在信使 RNA（mRNA）剪接和核糖体生物发生过程中重塑蛋白-RNA 复合物。该解旋酶的活性和 ATP 转换内在较低，并且在特定的细胞环境中通过 G-补丁（gp）因子被激活。gp 基序将解旋酶核心与灵活的 C 端结构域连接起来，但尚不清楚这如何影响催化过程中 RecA 结构域的运动以及 RNA 底物的解旋。我们开发了单分子Förster 共振能量转移（smFRET）报告器，以实时研究 Prp43 内 RecA 结构域的运动。没有 Pfa1（gp）时，这些结构域彼此靠近，主要采用封闭构象。添加 Pfa1（gp）会诱导开放状态，与 RNA 相互作用时甚至更为普遍。在开放状态下，Prp43 与结合的核苷酸的接触减少，并显示出快速的腺苷二磷酸（ADP）释放，从而加速从弱（ADP）到强（apo）RNA 结合状态的转变。使用 smFRET 标记 RNA 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 非结合状态之间切换，而不是与 RNA 解离。ATP 与无蛋白酶的酶结合会诱导沿 RNA 的易位，产生融化近端 RNA 结构所需的解旋力。在 ATP 转换过程中，Pfa1（gp）刺激 RecA 结构域在打开和关闭状态之间交替。因此，在 ADP 状态下，易位速度比从底物解离更快，从而允许沿 RNA 进行连续运动。我们提供了 DEAH/RHA 解旋酶运动的机制模型，并揭示了 G-补丁蛋白对 Prp43 调节的原理。
