Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

Abstract

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.

When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding

“…The structure of the Hera RBD in complex with a RNA tetranucleotide reveals recognition of a GGXY stretch by the RRM (Steimer et al, 2013). The tetranucleotide straddles helix α1, and binds to the same side of the RRM as does the loop region of hairpin 92 to the YxiN RBD ( Figure 3C,E).…”

“…The overall fold of the RRM is unchanged in the RNA-bound form, with the exception of the flexible C-tail that was disordered in the apo structure, but becomes ordered in the RNA complex. The C-tail is important for high affinity RNA binding, and most likely interacts with duplex regions in the vicinity of the single-stranded region recognized by the RRM (Steimer et al, 2013). Thus, the Hera RBD appears to recognize two distinct elements in the Hera RNA substrate, a single-stranded GGXY stretch, and a nearby duplex.…”

“…Upper panels: front view, bottom panels: top view (relative to the depiction in panel A). Modified with permission from Steimer et al (2013) [Steimer, L., Wurm, J.P., Linden, M.H., Rudolph, M.G., Wohnert, J., and Klostermeier, D. (2013). Recognition of two distinct elements in the RNA substrate by the RNA binding domain of the T. thermophilus DEAD box helicase Hera.…”

“…From a superposition of the Hera model with structures of the helicase core of Vasa (Sengoku et al, 2006) and of the RBD bound to RNA (Steimer et al, 2013), it is evident that the RNA bound to the RBD and the core have opposite polarities, with their 5′-ends facing towards each other ( Figure 4B). Although both RNA binding sites are connected by an elongated patch of positive electrostatic potential, it is clear from this polarity that they do not simply bind to one contiguous strand of a larger RNA.…”

“…The helix 91 that is unwound by YxiN using model RNA substrates, highlighted by the red box, is pointing away from the RNA binding site of the helicase core (arrow). (B) Model for the full-length Hera dimer Klostermeier, 2013), showing the RNA bound to the helicase core and to the RRM of the RBD (Steimer et al, 2013). The model was constructed from the structures comprised of RecA_C and the DD ; PDB-ID 3eas), RecA_C, the DD, and the RBD PDB-ID 3i32), the RBD in complex with RNA (Steimer et al, 2013;PDB-ID 4i67), and a homology model for the closed helicase core (RecA_N, RecA_C), using the Vasa structure in complex with RNA and ADPNP (Sengoku et al, 2006; PDB-ID 2db3) as a template.…”