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

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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.