The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

Abstract

DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly.

The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

“…For example, the C-terminal domains of the splicing helicases CYT-19 and Mss116 mediates interactions with structured RNA (Grohman et al, 2007;Mohr et al, 2008). In contrast, the C-terminal domains of DbpA and YxiN specifically interact with a hairpin in ribosomal RNA (Diges and Uhlenbeck, 2001;Tsu et al, 2001;Kossen et al, 2002;Karginov et al, 2005;Wang et al, 2006), and the C-terminal domain of Hera provides high affinity for ribosomal RNA and RNase P RNA, among others (Morlang et al, 1999;Linden et al, 2008). The influence of domains flanking the helicase core on RNA binding and on DEAD box mechanism in general will be discussed in detail in 'Modulation of the helicase core activity by interacting partners and flanking domains'.…”

“…As a unique feature so far, this C-terminal domain is bipartite and consists of a dimerization motif , and an RBD that mediates high affinity interaction with ribosomal RNA fragments and RNase P RNA (Linden et al, 2008). Despite the lack of sequence similarity with YxiN, the Hera RBD folds into a modified RRM ).…”

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop

“…8-Oxo-ATP was tested as an energy source for Hera helicase activity ( Figure 1C). Although wild-type Hera displayed ATP-dependent RNA unwinding activity on a 32/ 9mer RNA substrate derived from 23S rRNA (Linden et al, 2008), no unwinding of RNA in the presence of 8-oxo-ATP was observed. By contrast, Hera_Q28E showed no unwinding activity, neither with ATP nor with 8-oxo-ATP as the energy source ( Figure 1C).…”

“…They are named according to the characteristic sequence of their Walker B motif, which is implicated in ATP binding. DEAD-box proteins share a common modular architecture ( Figure 1A): a helicase core of two flexibly connected RecA-like domains (RecA_N and RecA_C), which are sometimes flanked by additional domains that confer substrate binding specificity, mediate protein/protein interactions or may contribute to duplex separation (Tsu et al, 2001;Kossen et al, 2002;Grohman et al, 2007;Linden et al, 2008;Mohr et al, 2008;Del Campo et al, 2009). In addition, DEAD-box helicases can also form dimers Rudolph and Klostermeier, 2009) via dedicated dimerization domains, as has recently been found for the Thermus thermophilus at resistant NA-dependent TPase, Hera He R A (Morlang et al, 1999), a DEAD-box RNA helicase presumably involved in ribosome assembly and assembly of RNase P (Linden et al, 2008).…”

“…DEAD-box proteins share a common modular architecture ( Figure 1A): a helicase core of two flexibly connected RecA-like domains (RecA_N and RecA_C), which are sometimes flanked by additional domains that confer substrate binding specificity, mediate protein/protein interactions or may contribute to duplex separation (Tsu et al, 2001;Kossen et al, 2002;Grohman et al, 2007;Linden et al, 2008;Mohr et al, 2008;Del Campo et al, 2009). In addition, DEAD-box helicases can also form dimers Rudolph and Klostermeier, 2009) via dedicated dimerization domains, as has recently been found for the Thermus thermophilus at resistant NA-dependent TPase, Hera He R A (Morlang et al, 1999), a DEAD-box RNA helicase presumably involved in ribosome assembly and assembly of RNase P (Linden et al, 2008). Upon cooperative binding of RNA and ATP (or an ATP analog), the helicase core collapses to form a composite RNA binding site traversing both RecA-like domains, as exemplified by crystal structures of helicase/ RNA complexes (Andersen et al, 2006;Bono et al, 2006;Sengoku et al, 2006;von Moeller et al, 2009).…”