Mechanism and regulation of RNA unwinding by DEAD-box RNA helicases

在RNA代谢的几乎所有过程中，RNA解旋酶利用ATP水解的能量来分离RNA双链。在DEAD-box解旋酶中，一个保守的解旋酶核心赋予基本的RNA解旋活性。双相分离与核苷酸驱动的解旋酶核在开放构象和封闭构象之间的交替有关。解旋酶核心的核苷酸结合和水解、RNA结合和解绕活性由解旋酶核心两侧的区域、低聚解旋酶中的其他原聚物以及相互作用伙伴调节，但其潜在机制在很大程度上是未知的。在本项目中，我们将研究解旋酶核心活性的调控机制。我们将使用枯草芽孢杆菌YxiN和嗜热芽孢杆菌Hera这两种包含c端rna结合域（RBD）的解旋酶作为特异性和非特异性解旋酶的代表。在前期研究成果的基础上，我们将进一步研究RNA与RBD结合后解旋酶核心的变构激活机制。通过定点诱变、atp酶和解绕实验以及单分子FRET实验，我们将剖析从RBD到解旋酶核心的通讯链，该通讯链介导了YxiN和Hera的核心激活。我们将进一步研究二聚解旋酶Hera在体内的解旋作用和机制。为了确定Hera在嗜热t菌中起作用的功能背景，我们在eCLIP-seq实验中确定了Hera的生理结合伙伴。Hera是唯一的二聚体DEAD-box解旋酶。我们将在单分子FRET实验中研究Hera二聚体中两个解旋酶核心在RNA解绕过程中的功能合作，使用最小的RNA底物来定义RNA结合和解绕的热力学和动力学框架，以及可以同时接触两个解旋酶核心的生理RNA底物。为了了解翻译起始过程中eIF4A活性的调控机制，我们将研究其他翻译起始因子和翻译高度依赖eIF4A的mrna的5 ' - utr对eIF4A构象周期动力学及其解绕活性的影响。为此，我们建立了一个体外翻译系统，使我们能够将翻译效率与eIF4A构象动力学和解绕联系起来。这些研究将揭示DEAD-box蛋白解旋酶核心通过附加结构域、其他原蛋白和相互作用伙伴调控的分子基础，并将进一步了解解旋酶核心活性如何适应特定解旋酶的体内功能。

RNA helicases use the energy of ATP hydrolysis to separate RNA duplexes in virtually all processes in RNA metabolism. In DEAD-box helicases, a conserved helicase core confers basal RNA unwinding activity. Duplex separation is linked to the nucleotide-driven alternation of the helicase core between open and closed conformations. The nucleotide binding and hydrolysis, RNA binding and unwinding activities of the helicase core are modulated by regions flanking the helicase core, by other protomers in oligomeric helicases, and by interaction partners, but the underlying mechanisms are largely unknown. In the proposed project, we will investigate the mechanisms of regulation of helicase core activities. We will use B. subtilis YxiN and T. thermophilus Hera, two helicases that comprise a C-terminal RNA-binding domain (RBD), as representatives for specific and non-specific helicases. Building on our results from the previous funding period, we will study the mechanism of allosteric activation of helicase core by RNA binding to the RBD. Using site-directed mutagenesis, ATPase and unwinding assays, and single-molecule FRET experiments, we will dissect the communication chain leading from the RBD to the helicase core that mediates core activation in YxiN and possibly in Hera. We will further investigate the in vivo role and the mechanism of unwinding by the dimeric helicase Hera. To define the functional context in which Hera acts in T. thermophilus, we have identified physiological binding partners of Hera in eCLIP-seq experiments. Hera is the only dimeric DEAD-box helicase. We will investigate the functional cooperation of the two helicase cores in the Hera dimer during RNA unwinding in single-molecule FRET experiments, using a minimal RNA substrate to define a thermodynamic and kinetic framework of RNA binding and unwinding, and a physiological RNA substrate that can contact both helicase cores simultaneously. To understand the regulatory mechanisms of eIF4A activity during translation initiation, we will study the effects of other translation initiation factors and of the 5’-UTRs of mRNAs whose translation shows a strong dependence on eIF4A on the kinetics of the eIF4A conformational cycle and its unwinding activity. To this end, we have established an in vitro translation system that allows us to correlate translation efficiencies with eIF4A conformational dynamics and unwinding. These studies will reveal the molecular basis for the regulation of the helicase core of DEAD-box proteins by additional domains, by other protomers, and by interaction partners, and will further our understanding of how the helicase core activity is tailored to the in vivo function of a particular helicase.