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

RNA分子对生命至关重要，但容易错误折叠。因此，在所有生物体中都发现了解决错误折叠的RNA结构和重塑蛋白质/RNA复合物的RNA解旋酶。ATP依赖性DEAD盒蛋白构成了一大类RNA解旋酶。它们由2个RecA样结构域组成，在apo状态下独立翻滚，但双链RNA（dsRNA）和ATP的结合导致形成具有二分RNA结合位点的刚性复合物。该复合物使RNA双链体不稳定，从而促进双链体解旋。关于双链RNA/ATP结合复合物和解旋循环的其他几种状态的结构信息仍然是难以捉摸的，这将大大增加我们对解旋过程的理解。杆菌我们将主要使用最先进的NMR技术和体外活性测定来解决两个主要问题：1。RecA结构域和RNA结合结构域之间的结构和功能相互作用：DbpA除了典型的RecA结构域外，还含有C末端RNA识别基序（RRM）。RRM与23 S rRNA的发夹92结合，并在核糖体生物发生期间将解旋酶核心锚定到肽基转移酶中心。rRNA与RRM的结合通过未知的机制强烈刺激DbpA的解旋活性。C-末端RecA结构域和RRM之间的相互作用已被证明。我们将求解包含两个结构域的结构。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，我们还将对野生型和DbpA基因敲除菌株的23 SrRNA进行体内结构探测实验，以确定DbpA解旋活性的靶位点. DEAD盒解旋酶双链体解旋的分子机制：在这里，我们将使用DbpA作为模型系统，以深入了解DEAD盒解旋酶的解旋机制。我们将在解旋循环的不同状态下稳定DbpA，并使用NMR和/或X射线晶体学分析这些状态。我们的主要重点将放在双链RNA/ATP结合的复合物，以阐明如何实现双链体不稳定。这些实验将产生前所未有的洞察DbpA和DEAD盒解旋酶的解旋周期期间的结构和动态变化。

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.