RNA binding and unwinding by the DEAD box protein Hera from Thermus thermorphilus

DEAD-box helicases are required in almost every step of RNA metabolism. Their common structural signature is a conserved helicase core, often flanked by N- or C-terminal extensions. The Thermus thermophilus DEAD box helicase Hera is built up modularly; it consists of the helicase core, and a C-terminus containing the dimerization domain (DD) and RNA binding domain (RBD)^[1]. Different RNAs such as 23S RNA and RNase P RNA can stimulate the Hera ATPase activity and induce a closed conformation of the core upon binding.^[2] Tight binding of the RNA substrate is promoted by the RBD. The RBD has an unusual RNA recognition motif (RRM) ^[2]. The binding mode of RNA to this motif in Hera is unknown. To identify the RNA binding site on the Hera RBD, aromatic and positively charged residues were replaced by alanines, and the RNA affinities were determined in anisotropy titrations. Our results suggest that binding of the RNA substrate is promoted by residues located on various structural elements of the RRM, including residues on the central β-strands, the α-helix as well as on the C-terminal loop and an internal loop. Unwinding of double-stranded RNA regions occurs at the catalytically active core domain after binding of RNA to the RBD. For the biochemical characterization of this process we established a FRET-based unwinding assay that allows following individual steps throughout the catalytic cyle of Hera. In combination with the conformational information already available new insights into the mechanism of duplex destabilization by Hera can be gained.