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.

Figure 1.

Hera constructs and sequence alignments. (A) Sequence alignment of the CTDs of T. thermophilus Hera, E. coli DbpA, and B. subtilis YxiN. The alignment was created with ClustalW. The CTD is highlighted in light green. Conserved positively charged residues are highlighted in red, positively charged residues in the Hera CTD are marked in green. The green bars indicate the N-terminal, center- and C-terminal regions with a slight clustering of positively charged residues in the Hera CTD. (B) Sequence comparison of the RNR-motif in RnpA from E. coli, B. subtilis and T. thermophilus with the RNase P motif in Hera. The three conserved arginines in the RNR motif are marked in red.

Figure 2.

RNA-induced conformational changes in Hera. FRET histograms for Hera_E115C/R260C in the absence of RNA (top panels), in the presence of RNA (center panels) and in the presence of the respective RNA and ADPNP (bottom panels). (A) PolyU-RNA, (B) 153-mer rRNA and (C) RNase P RNA. The broken gray line marks the FRET efficiency for Hera. An increase in FRET efficiency from 0.65 to 0.8 is detected in the presence of all three RNAs and ADPNP (gray arrows), indicating a closure of the cleft within the Hera helicase domain in response to RNA and ADPNP binding. (D) Overview of FRET efficiencies for Hera_E115C/R260C in the presence of different nucleic acid substrates. Open squares: in the presence of nucleic acid only, closed squares: in the presence of nucleic acid and ADPNP. In the context of full-length Hera, polyU-RNA, RNase P RNA and the ribosomal RNA fragments induce a closure of the inter-domain cleft in the helicase core (circled).

Figure 3.

RNA-induced conformational changes in Hera_core. FRET histograms for Hera_core_E115C/R260C in the absence and presence of RNA. (A) No RNA, (B) polyU-RNA and ADPNP, (C) 153-mer and ADPNP and (D) RNase P RNA and ADPNP. The FRET efficiency of 0.50 in the absence of ligands (marked by the broken gray line) indicates a less compact conformation of the core compared to full-length Hera (E_FRET = 0.65). Only polyU-RNA induces the formation of the closed conformer (E_FRET = 0.75), whereas the FRET efficiency remains at 0.50 in the presence of the 153-mer or RNase P RNA, indicating that these RNAs do not elicit a conformational change in Hera_core.

Figure 4.

ATPase stimulation of Hera by nucleic acid substrates. Hera is a Michaelis–Menten enzyme. The intrinsic ATPase activity is characterized by a k_cat value of 7 × 10⁻³ s⁻¹. RNase P RNA (squares), the 153-mer (open circles) and polyU-RNA (open triangles) stimulate the intrinsic ATPase activity of Hera. The Michaelis–Menten constants are: k_cat = 1.8 s⁻¹, K_M,app = 77 nM (29 μM nucleotides, RNase P RNA), k_cat = 1.3 s⁻¹, K_M,app = 390 nM (59 μM nucleotides, 153-mer) and k_cat = 2.6 s⁻¹, K_M,app = 134 μM (nucleotides, polyU-RNA), corresponding to a 190- to 370-fold ATPase stimulation.

Figure 5.

No competition of RnpA and Hera for RNase P RNA. (A) FRET histograms for Hera_E115C/R260C, 50 nM unlabeled Hera, 50 nM RNase P RNA and 3 mM ADPNP in the absence (upper panel) and in the presence (lower panel) of E. coli RnpA (1 μM). (B) The FRET efficiency in the absence of RnpA (marked by the gray broken line) is indicative of the Hera/RNase P RNA/ADPNP complex with a closed conformation of the helicase core. It is unaffected by the addition of RnpA, indicating that RnpA does not compete with Hera for RNase P RNA. (B) FRET histograms for Hera_E115C/R260C and 3 mM ADPNP in the absence (upper panel) and in the presence (lower panel) of preformed RnpA/RNase P RNA complex (50 nM RNase P RNA, 1 µM RnpA). The FRET efficiency in the absence of RnpA/RNase P (gray broken line) indicates the open conformation. Upon addition of the preformed RnpA/RNase P complex, Hera binds to the RNase P RNA and adopts the closed conformation.

Figure 6.

No interaction of RNase P RNA with B. subtilis YxiN. FRET histograms for YxiN_C61/267A_A115/S229C in the absence of ligands (upper panel) and in the presence (bottom panel) of RNase P RNA and ADPNP. The FRET efficiency of ∼0.3 in the absence of RNase P RNA is indicative of the open YxiN conformation (21) and remains unchanged upon addition of RNase P RNA.

Figure 7.

The RNase P motif mutant still interacts with RNase P RNA and the ribosomal 153-mer. (A) FRET histograms of Hera_Rpm_E115C/E260C in the absence of nucleic acids, and in the presence of 1 mM polyU-RNA, 400 nM of the 153-mer ribosomal RNA fragment, or 400 nM RNase P RNA and 3 mM ADPNP. All RNAs induce a conformational change in Hera_Rpm, demonstrating that the RNase P motif in Hera is not required for the interaction with these RNA substrates. The FRET efficiency of the open conformer is marked by a gray broken line. (B) RNA stimulation of the Hera_Rpm steady state ATPase activity by RNase P RNA (squares) and the 153-mer (open circles). The Michaelis–Menten parameters are k_cat = 1.0 s⁻¹, K_M,app = 26 nM (10 μM nucleotides, RNase P RNA), k_cat = 1.4 s⁻¹ and K_M,app = 210 nM (32 μM nucleotides, 153-mer). Hence, the ATPase activity of the Hera mutant carrying three mutations in the putative RNase P motif is still efficiently stimulated by RNase P RNA and the 153-mer.

Figure 7.

The RNase P motif mutant still interacts with RNase P RNA and the ribosomal 153-mer. (A) FRET histograms of Hera_Rpm_E115C/E260C in the absence of nucleic acids, and in the presence of 1 mM polyU-RNA, 400 nM of the 153-mer ribosomal RNA fragment, or 400 nM RNase P RNA and 3 mM ADPNP. All RNAs induce a conformational change in Hera_Rpm, demonstrating that the RNase P motif in Hera is not required for the interaction with these RNA substrates. The FRET efficiency of the open conformer is marked by a gray broken line. (B) RNA stimulation of the Hera_Rpm steady state ATPase activity by RNase P RNA (squares) and the 153-mer (open circles). The Michaelis–Menten parameters are k_cat = 1.0 s⁻¹, K_M,app = 26 nM (10 μM nucleotides, RNase P RNA), k_cat = 1.4 s⁻¹ and K_M,app = 210 nM (32 μM nucleotides, 153-mer). Hence, the ATPase activity of the Hera mutant carrying three mutations in the putative RNase P motif is still efficiently stimulated by RNase P RNA and the 153-mer.

Figure 8.

RNA unwinding by Hera, Hera_core and Hera_Rpm. A total of 5 μM of the 32/9-mer rRNA were incubated with 10 μM Hera, Hera_core or Hera_Rpm in the absence or presence of 5 mM ATP at 25°C for 30 min, and products were separated on a 15% polyacrylamide gel. Hera unwinds ∼90% of the substrate in the presence of ATP, whereas no unwinding is observed in the absence of ATP. Hera_core is not able to unwind this RNA. Hera_Rpm shows ATP-dependent RNA unwinding activity with the same extent (90%) as wild-type Hera. The RNase P motif is thus dispensable for the helicase activity of Hera.