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

在RNA新陈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，保守的解旋酶核心赋予基本的RNA解旋活性。双链分离与核苷酸驱动的解旋酶核心在开放和闭合构象之间的交替有关。解旋酶核心的核苷酸结合和水解、RNA结合和解旋活性受解旋酶核心两侧区域、寡聚解旋酶中的其他原基和相互作用伙伴的调节，但其潜在的机制在很大程度上是未知的。在拟议的项目中，我们将研究解旋酶核心活性的调节机制。我们将使用枯草杆菌YxiN和嗜热杆菌Hera这两个包含C末端RNA结合域(RBD)的解旋酶作为特定和非特异性解旋酶的代表。基于我们在前一个资助时期的研究结果，我们将研究通过RNA与RBD结合来激活解旋酶核心的变构机制。利用定点突变、ATPase和解离分析以及单分子FRET实验，我们将剖析从RBD到解旋酶核心的通信链，该核心在YxiN中介导核心激活，在HERA中也可能如此。我们将进一步研究二聚体解旋酶HERA在体内的作用和解离机制。为了确定HERA在嗜热杆菌中发挥作用的功能背景，我们在eCLIP-SEQ实验中确定了HERA的生理结合伙伴。Hera是唯一的二聚体死盒解旋酶。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解旋过程中的功能协作，使用最小的RNA底物来定义RNA结合和解离的热力学和动力学框架，以及一个可以同时接触两个解旋酶核心的生理RNA底物。为了了解eIF4A活性在翻译启动过程中的调节机制，我们将研究其他翻译启动因素以及翻译强烈依赖eIF4A的mRNAs的5‘-UTRs对eIF4A构象循环及其解离活性的动力学的影响。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构象动力学和解离联系起来。这些研究将揭示DEAD-box蛋白解旋酶核心受其他结构域、其他原基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是如何根据特定解旋酶在体内的功能而定制的。

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.