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

RNA分子是生命所必需的，但容易发生错误折叠。因此，在所有生物体中都发现了RNA解旋酶，它可以分解错误折叠的RNA结构，重塑蛋白质/RNA复合体。依赖于ATP的DEAD盒蛋白构成了一大类RNA解旋酶。它们由2个在apo状态下独立翻滚的RecA样结构域组成，但双链RNA(DsRNA)与ATP的结合导致形成具有两部分RNA结合位点的刚性复合体。这种复合体破坏了RNA双链的稳定，从而促进了双链的解离。关于dsRNA/ATP结合复合体和解离周期的其他几个状态的结构信息仍然难以捉摸，这将极大地增加我们对解离过程的理解。在这项提议中，我们旨在提供对大肠杆菌死盒解旋酶DbpA双重解离的结构和动力学基础的见解。我们将主要利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：1.RecA结构域和RNA结合域之间的结构和功能相互作用：DbpA除了标准的RecA结构域外，还包含一个C末端RNA识别基序(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转移酶中心。RRNA与RRM的结合以一种未知的机制强烈刺激DbpA的解离活性。C-末端RecA结构域和RRM之间的相互作用已经被证明。我们将解决包含这两个域的构造的结构。为了深入了解DbpA的RNA依赖激活，我们将研究RNA结合对DbpA结构和动力学的影响。此外，我们还将对野生型和DbpA基因敲除菌株中的23S rRNA进行体内结构探测实验，以确定DbpA解离活性的靶点。双链解链的分子机制：在这里，我们将以DbpA为模型系统，深入了解死盒解旋酶的解链机制。我们将在解卷周期的不同状态下稳定DbpA，并使用核磁共振和/或X射线结晶学分析这些状态。我们的主要重点将放在dsRNA/ATP结合的复合体上，以阐明双链失稳是如何实现的。这些实验将对DbpA和死盒解旋酶在解旋周期中的结构和动态变化产生前所未有的见解。

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.