LAUSR

DEAD-box proteins catalyze the ATP-dependent unwinding of RNA duplexes. Their common helicase core, formed by two RecA-like domains, is typically flanked by additional domains that modulate helicase function by hitherto unidentified mechanisms. To understand the regulation of the DEAD-box helicase YxiN by its C-terminal RNA binding domain (RBD), we investigated the effect of RNA binding to the RBD on its position relative to the core. To this end, we used distances from single molecule FRET experiments on freely diffusing YxiN molecules by confocal microscopy as restraints to model structures of YxiN in the absence and presence of RNA. RNA binding to the RBD causes a substantial movement of the RBD, from a position close to the C-terminal RecA-domain of the core towards the N-terminal RecA-domain. This movement is independent of direct contacts of the bound RNA with the core. Importantly, RNA binding to the RBD is communicated to the core, and stimulates ATP hydrolysis and RNA unwinding activities. RNA unwinding by DEAD-box helicases has been linked to a conformational cycle, involving switching of the helicase core between open and closed conformations. In single molecule FRET experiments on immobilized YxiN using total internal reflection microscopy, we investigated the conformational dynamics of the YxiN core when RNA is bound to the RBD. We show that the conformational space of the helicase core, and hence the effect of the RNA on core activities, depends on the identity of the bound RNA, pointing to a differential recognition of RNA elements in their structural context. Our results establish that the RBD does not merely act as a passive anchor for YxiN on the RNA substrate but instead actively modulates the functions of the helicase core. Regulation of core activities by RNA-induced movement of ancillary domains may constitute a general regulatory mechanism of DEAD-box protein activity.