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

DEAD-box RNA解旋酶对RNA解旋的机制和调控

基本信息

批准号：
250786717
负责人：
Professorin Dr. Dagmar Klostermeier
金额：
--
依托单位：
Institut für Physikalische Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/250786717?language=en
关键词：
Mechanism regulation RNA unwinding DEAD

项目摘要

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.

RNA解旋酶在RNA代谢的几乎所有过程中使用ATP水解的能量来分离RNA双链体。在DEAD盒解旋酶中，保守的解旋酶核心赋予基础RNA解旋活性。双链体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侧翼区域、寡聚解旋酶中的其他原聚体和相互作用伴侣的调节，但基本机制在很大程度上是未知的。本研究将探讨解旋酶核心活性的调控机制。我们将使用B。subtilis YxiN和T.嗜热菌Hera的两种解旋酶，其包含C末端RNA结合结构域（RBD），作为特异性和非特异性解旋酶的代表。基于我们上一个资助期的结果，我们将研究RNA结合RBD对解旋酶核心的变构激活机制。使用定点诱变，ATP酶和解旋测定，和单分子FRET实验，我们将解剖的通信链导致从RBD的解旋酶核心介导的核心激活YxiN和可能在赫拉。我们将进一步研究在体内的作用和解旋的二聚体解旋酶赫拉的机制。定义赫拉在T中的作用的功能语境。在嗜热菌中，我们已经在eCLIP-seq实验中鉴定了Hera的生理结合配偶体。Hera是唯一的二聚DEAD盒解旋酶。我们将调查的两个解旋酶核心的赫拉二聚体在RNA解旋过程中在单分子FRET实验的功能合作，使用最小的RNA基板，以定义一个热力学和动力学框架的RNA结合和解旋，和生理RNA基板，可以同时接触两个解旋酶核心。为了理解翻译起始过程中eIF 4A活性的调控机制，我们将研究其他翻译起始因子和其翻译对eIF 4A具有强烈依赖性的mRNA的5 '-UTR对eIF 4A构象循环动力学及其解旋活性的影响。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eIF 4A构象动力学和解旋联系起来。这些研究将揭示由额外的结构域，由其他原聚体，并通过相互作用的合作伙伴的解旋酶核心的DEAD盒蛋白质的调节的分子基础，并将进一步我们的解旋酶核心活性是如何定制一个特定的解旋酶的体内功能的理解。