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.

RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for non-sequence-specific interaction with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not defined. The DEAD-box helicase Hera from Thermus thermophilus contains a helicase core, followed by a dimerization domain and an RNA binding domain that folds into an RNA recognition motif (RRM). The RRM mediates high affinity binding to an RNA hairpin, and an adjacent duplex is then unwound by the helicase core. Hera is a cold-shock protein, and has been suggested to act as an RNA chaperone under cold-shock conditions. Using crosslinking immunoprecipitation of Hera/RNA complexes and sequencing, we show that Hera binds to a large fraction of T. thermophilus RNAs under normal-growth and cold-shock conditions without a strong sequence preference, in agreement with a structure-specific recognition of RNAs and a general function in RNA metabolism. Under cold-shock conditions, Hera is recruited to RNAs with high propensities to form stable secondary structures. We show that selected RNAs identified, including a set of tRNAs, bind to Hera in vitro, and activate the Hera helicase core. Gene ontology analysis reveals an enrichment of genes related to translation, including mRNAs of ribosomal proteins, tRNAs, tRNA ligases, and tRNA-modifying enzymes, consistent with a key role of Hera in ribosome and tRNA metabolism.