Supplementary Figure 2: An excess of HflX splits 70S ribosomes in vitro and in vivo.
From: HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions
(a-b) The splitting of purified 70S (0.3 ÎĽM) (a) or crude 70S ribosome in cell lysate (freshly prepared by ultra-sonication) (b), with 20-fold excess of HflX in the absence (Apo) or presence of different nucleotides (GMPPNP, GTP, and GDP, 1 mM) was checked by SDGC. (c) Overexpression of HflX from the hflX-pBAD plasmid with 1% L-arabinose resulted in slower growth rate of the bacterial host E. coli BW25113 (WT). The vector alone was treated similarly as a control. Data shown are means and s.d. (n= 3 cell-culture replicates). (d) Polysome profile of the mid-log phase E. coli BW25113 cells without (0%) and with induction of HflX by adding 1 % or 2 % (w/v) L-arabinose (L-ara). While HflX overexpression leads to the increase in the 50S fraction. (e) SDS-PAGE and western blotting analysis (anti-his) to check the expression level of recombinant HflX in the above cells. P, empty pBAD vector; H, pBADhflX plasmid. The bands corresponding to HflX are indicated as asterisks.
