Fig. 2
From: RNA helicases mediate structural transitions and compositional changes in pre-ribosomal complexes
The ATPase activity of Mak5 and Spb4 is required for LSU biogenesis. a Schematic view of the domains of Mak5 and Spb4. The relative positions of the conserved RecA-like domains, and DEAD and SAT motifs, are highlighted. Amino acid numbers of domain boundaries are indicated above and amino acids substitutions introduced are marked below. b His10-ZZ-tagged wild-type Mak5 (Mak5WT), Mak5DEAD, Mak5SAT, wild-type Spb4 (Spb4WT), Spb4DEAD, Spb4SAT were purified from E. coli, separated by SDS-PAGE and visualised by Coomassie staining. c, d The ATPase activity of recombinant Mak5 (c), Spb4 (d) and their mutants in the presence of different concentrations of RNA was monitored using NADH-coupled ATPase assays. Data from three independent experiments is shown as mean ± standard deviation. e The pTetO7-HA-MAK5–derived yeast strains carrying an empty pRS415 plasmid (EV) or pRS415-based plasmids for expression of Mak5WT, Mak5DEAD or Mak5SAT were grown in YPD before 10-fold serial dilution and growth on either YPD (-dox) or YPD supplemented with doxycycline (+dox) plates. Growth was recorded after 72 h growth at 30 °C. f Growth of the pGAL1-HA-SPB4 yeast strain transformed with pRS415 (EV) or plasmids for expression of Spb4WT, Spb4DEAD or Spb4SAT was analysed on YPG (Gal) or YPD (Glu) as in (e). g, h The pTetO7-HA-MAK5 and pGAL1-HA-SPB4 complementation strains were grown as in (e, f) before harvesting. RNAs were isolated, separated by agarose-glyoxal gel electrophoresis and pre-rRNAs were detected by northern blotting using probes hybridising in ITS1 and ITS2. Mature rRNAs were detected by methylene blue staining. The experiments presented in (e–h) were all performed in triplicate and representative data is shown
