Mechanism and regulation of RNA unwinding by DEAD-box RNA helicases

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RNA helicases use the energy of ATP hydrolysis to separate RNA duplexes in virtually all processes in RNA metabolism. In DEAD-box helicases, a conserved helicase core confers basal RNA unwinding activity. Duplex separation is linked to the nucleotide-driven alternation of the helicase core between open and closed conformations. The nucleotide binding and hydrolysis, RNA binding and unwinding activities of the helicase core are modulated by regions flanking the helicase core, by other protomers in oligomeric helicases, and by interaction partners, but the underlying mechanisms are largely unknown. In the proposed project, we will investigate the mechanisms of regulation of helicase core activities. We will use B. subtilis YxiN and T. thermophilus Hera, two helicases that comprise a C-terminal RNA-binding domain (RBD), as representatives for specific and non-specific helicases. Building on our results from the previous funding period, we will study the mechanism of allosteric activation of helicase core by RNA binding to the RBD. Using site-directed mutagenesis, ATPase and unwinding assays, and single-molecule FRET experiments, we will dissect the communication chain leading from the RBD to the helicase core that mediates core activation in YxiN and possibly in Hera. We will further investigate the in vivo role and the mechanism of unwinding by the dimeric helicase Hera. To define the functional context in which Hera acts in T. thermophilus, we have identified physiological binding partners of Hera in eCLIP-seq experiments. Hera is the only dimeric DEAD-box helicase. We will investigate the functional cooperation of the two helicase cores in the Hera dimer during RNA unwinding in single-molecule FRET experiments, using a minimal RNA substrate to define a thermodynamic and kinetic framework of RNA binding and unwinding, and a physiological RNA substrate that can contact both helicase cores simultaneously. To understand the regulatory mechanisms of eIF4A activity during translation initiation, we will study the effects of other translation initiation factors and of the 5’-UTRs of mRNAs whose translation shows a strong dependence on eIF4A on the kinetics of the eIF4A conformational cycle and its unwinding activity. To this end, we have established an in vitro translation system that allows us to correlate translation efficiencies with eIF4A conformational dynamics and unwinding. These studies will reveal the molecular basis for the regulation of the helicase core of DEAD-box proteins by additional domains, by other protomers, and by interaction partners, and will further our understanding of how the helicase core activity is tailored to the in vivo function of a particular helicase.