Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California, USA.
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
J Biol Chem. 2022 Oct;298(10):102368. doi: 10.1016/j.jbc.2022.102368. Epub 2022 Aug 11.
During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.
在翻译起始过程中，真核起始因子 (eIF) 4E、eIF4A 和 eIF4G 成分协调其结合活性以调节 eIF4F 与 mRNA 结合的基本机制尚未完全确定。在这里，我们使用荧光各向异性为未加帽 RNA 与人 eIF4F 的相互作用生成热力学和动力学框架。我们证明 eIF4E 与 eIF4G 中的自动抑制结构域的结合生成了 eIF4F 复合物与 RNA 的高亲和力结合构象。此外，我们表明 eIF4A 成分的核苷酸结合状态进一步调节 eIF4F 对未加帽 RNA 的结合，在存在与不存在 ATP 的情况下观察到平衡解离常数降低了四倍。实时监测未加帽 RNA 的解离揭示了 ATP 将 RNA 与 eIF4F 的解离速率常数降低了约 4 个数量级。因此，eIF4A 从 eIF4F 释放 ATP 将 eIF4F 置于具有非常快速的 RNA 结合和解离速率的动态状态。监测 eIF4A 与 eIF4G 的结合动力学框架揭示了两个不同的速率常数，这可能反映了 eIF4F 复合物的两种构象状态。此外，我们确定 eIF4G 自动抑制结构域促进了更稳定、动态性更小的 eIF4A 结合状态，该状态被 eIF4E 结合所克服。总体而言，我们的数据支持这样一种模型，即 eIF4E 与 eIF4G/4A 的结合稳定了 eIF4F 的高亲和力 RNA 结合状态，并使 eIF4A 能够与 eIF4G 形成更具动态性的相互作用。这种动态构象可能有助于 eIF4F 在扫描过程中快速结合和释放 mRNA。
