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. 2023 Jan 31;51(3):1326–1352. doi: 10.1093/nar/gkad033

Figure 4.

Figure 4.

DEAD box RNA helicases accept 3′-tracer tRNAs as substrates. (A) Fixed-time point activity assays using increasing concentrations of recombinant MBP-DDX3X, MBP-DDX5-GST, eIF4A1-GST dialysed in their respective (canonical) RNA helicase buffer and 3′-tracer tRNAs (8 nM final) in the presence of equimolar ATP/MgCl2 (2 mM). As controls, the highest protein concentration was incubated with 3′-tracer tRNAs without ATP. To control for 3′-tracer tRNA and 3′ tsRNA migration, 3′-tracer tRNAs were sampled before and after heat denaturation (Input). Reactions were separated by nPAGE and 32P-signals were collected as described above. Black arrowhead, 3′-tracer tRNAs; green arrowhead, 3′ tsRNAs. (B) Fixed-time point activity assays as in (A) using increasing concentrations of recombinant His-DDX1 and 3′-tracer tRNAs in canonical RNA helicase buffer. Description of Figure details as in (A). (C) Fixed-time point activity assays as in (A) using increasing concentrations of recombinant GST-DHX36136-988 and 3′-tracer tRNAs in canonical RNA helicase buffer. Description of Figure details as in (A). (D) Fixed-time point activity assays using increasing concentrations of recombinant MBP-DDX3X, MBP-DDX5-GST, eIF4A1-GST and 3′-tracer tRNAs (8 nM final) in the presence of equimolar ATP/MgCl2 (2 mM), but in non-canonical RNA helicase buffer (buffer d, see Supplementary Figure S1G). As controls, the highest protein concentration was incubated with 3′-tracer tRNAs without ATP. Description of figure details as in (A).