Inter-domain communication in the dimeric DEAD-box helicase Hera from T. thermophilus and implications for the mechanism of RNA unwinding.

The Thermus thermophilus DEAD-box helicase Hera consists of the conserved helicase core, followed by a dimerization domain (DD) and an RNA-binding domain (RBD). The RBD mediates high-affinity binding to an RNA hairpin; the DD mediates formation of a stable dimer. In the dimer, the active sites of the two helicase cores face each other in an ideal configuration to cooperate functionally in RNA unwinding. Here, we dissect the communication between the two RBDs and helicase cores by characterizing dimeric deletion variants with two cores, but two, one, or no RBDs, variants with both RBDs, but two, one, or no functional core, and variants with one core and one RBD, either on the same or opposite protomers. We show that RNA binds to Hera in a two-step mechanism, with an initial interaction between the RBD and a hairpin, followed by the interaction of the core with the flanking single- or double-stranded region. The duplex preferentially interacts with the core on the same protomer in the absence of ATP, but in the presence of ATP, interactions with the other core become possible. Overall, our results point to limited but significant cooperativity between the two protomers in RNA unwinding.