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

Basic data for this project

Description

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 alternation of the helicase core between open and closed conformations in the nucleotide cycle, linked to switches in RNA affinity. The translational initiation factor eIF4A is a minimal DEAD-box protein that consists of a helicase core only. Its activity is up-regulated by a number of other translation initiation factors, but the underlying mechanisms are ill-defined. Regions flanking the helicase core are implicated in nucleotide binding and hydrolysis, binding of RNA and protein partners, and duplex destabilization and unwinding by largely unknown mechanisms.In the proposed project, we will investigate the regulation of the helicase core by additional domains and by interaction partners. We will study the functional cooperation of RBDs with the helicase core, using B. subtilis YxiN and T. thermophilus Hera as representatives for specific and non-specific helicases. In both helicases, a C-terminal RNA-binding domain binds to RNA and presents adjacent duplexes to the helicase core. We will follow RNA-induced movement of the RBDs relative to the helicase core in the nucleotide cycle in single molecule-FRET experiments. The pathway of bound RNA substrates from the RBD to the active site for unwinding on the helicase core will be defined in mutagenesis/binding studies. We will further investigate the mechanism of unwinding and the in vivo role of Hera. To define the functional context in which Hera acts in T. thermophilus, in vivo interaction partners (physiological RNA substrates and protein partners) will by identified by deep sequencing and mass-spectrometry approaches. A possible function of Hera as a cold-shock protein will be tested directly. Hera is the only dimeric DEAD-box helicase, and we will investigate the functional cooperation of the two helicase cores in the Hera dimer during RNA unwinding in single molecule FRET experiments. To understand the regulatory mechanisms of eIF4A activity, we will study the effects of other translation initiation factors on the kinetics of the eIF4A conformational cycle in single molecule-FRET experiments by TIRF-microscopy. We will directly test a working model that these factors differentially affect individual steps in the catalytic cycle. Ultimately, we will establish single molecule-FRET experiments in an in vitro translation system to study the molecular basis of regulation for eIF4A and different helicases implicated in translation initiation.These studies will reveal the molecular basis for the regulation of the helicase core of DEAD-box proteins by flanking domains, by multiple protomers, and by additional interaction partners, and will further our understanding of how the helicase core activity is tailored to the in vivo function of a particular helicase.