Structural And Functional Insights Into The Dimeric Escherichia Coli Cold Shock DEAD-box Helicase CsdA

DEAD-box proteins belong to a ubiquitous family of RNA helicases, which are widely found from prokaryotes to eukaryotes and participate in multiple cellular processes, including pre-mRNA splicing, translation initiation, modulating RNA-protein complexes, RNA decay and ribosome biogenesis. DEAD-box proteins of helicase superfamily II consist of a helicase core with two canonical tandem RecA-like domains that contain several conserved motifs, one of them is D-E-A-D motif, which provide RNA and ATP binding sites to remodel high-order structures of RNAs and RNA/protein complexes. In addition, DEAD-box proteins contain auxiliary extensions in their N/C-terminus, which are thought to be essential to their specific recognition and interaction with different RNA or proteins. DEAD-box RNA helicases display both ATPase and unwinding activities.There are five DEAD-box RNA helicases, CsdA, DbpA, SrmB, Rh1E and Rh1B,in E. coli. CsdA is associated with the pre50S particles and is involved in the biogenesis of the 50S subunit; and plays an essential role in the survival of E. coli at low temperature, where strains with the deletion of the gene of CsdA protein results in a deficit in the biogenesis of the 50S subunit and an accumulation of 40S particles corresponds to incompletely assembled 50S subunit. In addition, the overexpression of CsdA can stabilize inefficiently translated mRNA in E. coli and recent study demonstrated that in E. coli at low temperature, the translation of rpoS mRNA that encodes stationary-phase and general stress response factor RpoS was not only regulated by the small RNA DsrA and the RNA chaperon Hfq but was also activated by CsdA.The csdA gene encodes a multi-domain protein of 629 amino acids. Sequence alignments show that CsdA has a conserved core of -365 amino acids and a long uncharacterized C-terminal region. However, the constitution of its C-terminal regions and the structure of CsdA are currently unclear. Here, we showed that CsdA not only consists of two tandem RecA-like domains but also contains previously uncharacterized dimerization domain and RNA binding domain ?RBD?. We determined the structures of all domains by X-ray crystallography or NMR. SAXS?small angle X-ray scattering? and 19F-PREs ?19F based paramagnetic relaxation enhancement? experiments revealed the conformational rearrangements of the helicase core of CsdA and structural model of the truncated CsdA₅64 was built.Biochemical experiments suggested DD is indispensable for the stability of CsdA.Our studies for the first time demonstrated that CsdA is a stable dimer at cold shock situations. The enzymatic assays and Fluorescence Polarization ?FP? experiments indicated the important roles of the C-terminal regions for binding RNAs. Moreover,CsdA_RBD could bind to a 32nt RNA ?containing a fragment of 23S rRNA H92? and prefer binding single-stranded G-rich RNAs, which was expected to bring helicase core to unwind the adjacent duplex.