Molecular mechanisms underlying RNA unwinding by the DEAD-box helicase DbpA

DEAD-box解旋酶DbpA解旋RNA的分子机制

• 批准号: 429252379
• 负责人: Professor Dr. Remco Sprangers, since 3/2022
• 金额: --
• 依托单位: Lehrstuhl für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/429252379?language=en
• 关键词: Molecular; mechanisms; underlying; RNA; unwinding

RNA molecules are essential for life, but prone to misfolding. RNA helicases that resolve misfolded RNA structures and remodel Protein/RNA complexes are therefore found in all organisms. The ATP-dependent DEAD box proteins constitute a large class of RNA helicases. They consist of 2 RecA like domains that tumble independently in the apo state, but binding of double stranded RNA (dsRNA) and ATP leads to the formation of rigid complex with a bipartite RNA binding site. This complex destabilizes the RNA duplex and thereby facilitates duplex unwinding. Structural information on the dsRNA/ATP-bound complex and on several other states of the unwinding cycle is still elusive and would greatly increase our understanding of the unwinding process.In this proposal we aim to provide insights into the structural and dynamical basis underlying duplex unwinding by the DEAD box helicase DbpA from E. coli. We will addresses two main questions using mainly state-of-the-art NMR techniques and in vitro activity assays: 1. The structural and functional interplay between the RecA domains and the RNA binding domain: DbpA contains a C-terminal RNA recognition motif (RRM) in addition to the canonical RecA domains. The RRM binds to hairpin 92 of the 23S rRNA and anchors the helicase core to the peptidyl transferase center during ribosome biogenesis. Binding of the rRNA to the RRM strongly stimulates the unwinding activity of DbpA by an unknown mechanism. An interaction between the C-terminal RecA domain and the RRM has been shown. We will solve the structure of a construct comprising both domains. To gain insights into the RNA dependent activation of DbpA we will then study the influence of RNA binding on the structure and dynamics of DbpA. In addition we will perform in vivo structure probing experiments on 23S rRNA in wildtype and DbpA knockout strains to identify the target sites of the DbpA unwinding activity.2. The molecular mechanism of duplex unwinding by DEAD box helicases: Here we will use DbpA as a model system to gain insights into the unwinding mechanism of DEAD box helicases. We will stabilize DbpA in the different states of the unwinding cycle and analyze these states using NMR and/or X-ray crystallography. Our main focus will be on the dsRNA/ATP-bound complex to elucidate how duplex destabilization is achieved. These experiments will yield unprecedented insights into the structural and dynamic changes during the unwinding cycle of DbpA and DEAD box helicases in general.

RNA分子对生命至关重要，但容易发生错误折叠。RNA解旋酶可以分解错误折叠的RNA结构并重塑蛋白质/RNA复合物，因此在所有生物体中都存在。依赖atp的DEAD box蛋白构成了一大类RNA解旋酶。它们由2个类似RecA的结构域组成，这些结构域在载脂蛋白状态下独立翻滚，但双链RNA （dsRNA）和ATP的结合导致刚性复合物与双链RNA结合位点形成。该复合物破坏RNA双链的稳定性，从而促进双链解绕。关于dsRNA/ atp结合复合物的结构信息和解绕周期的其他几个状态仍然难以捉摸，这将大大增加我们对解绕过程的理解。在这个提议中，我们的目的是提供对大肠杆菌中DEAD盒解旋酶DbpA双解绕的结构和动力学基础的见解。我们将主要使用最先进的核磁共振技术和体外活性分析来解决两个主要问题：RecA结构域和RNA结合结构域之间的结构和功能相互作用：DbpA除了包含典型的RecA结构域外，还包含c端RNA识别基元（RRM）。在核糖体生物发生过程中，RRM与23S rRNA的发夹92结合，并将解旋酶核心锚定在肽基转移酶中心。rRNA与RRM的结合通过一种未知的机制强烈刺激DbpA的解绕活性。c端RecA结构域与RRM之间存在相互作用。我们将解决包含两个域的构造的结构。为了深入了解DbpA的RNA依赖性激活，我们将研究RNA结合对DbpA结构和动力学的影响。此外，我们将对野生型和DbpA敲除菌株的23S rRNA进行体内结构探测实验，以确定DbpA解绕活性的靶位点。DEAD盒解旋酶双解旋的分子机制：本文将以DbpA为模型系统，深入了解DEAD盒解旋酶的解旋机制。我们将稳定DbpA在不同的解绕循环状态，并使用核磁共振和/或x射线晶体学分析这些状态。我们的主要重点将放在dsRNA/ atp结合的复合体上，以阐明如何实现双工不稳定。这些实验将对DbpA和DEAD解旋酶在解绕周期中的结构和动态变化产生前所未有的见解。