Rae1 cleavage leads to ribosome rescue by tmRNA. (A) Schematic of the gfp-S1025 fusion. The fusion is placed under the control of the IPTG-inducible Hyperspank promoter in vector pDR111. The Rae1 site within S1025 is indicated by red scissors. The position of the probes used is indicated by black bars. (B) Northern blot analysis at times after rifampicin addition strains containing the gfp-S1025 fusion and mutant ssrA (ssrA-H6DD) transformed either as a plasmid expressing the WT protein Rae1 (pRae1) or the empty vector. The blot was probed in a sequential order with oligos complementary to the 3′ UTR (CC429), gfp (CC2422), and then 16S rRNA (CC058) as a loading control. (C) Proteins were extracted from strains used in panel B, absorbed, washed and eluted from Ni2+-NTA agarose beads. The different fractions were assayed by western blot using GFP-specific antibodies. Fraction L corresponds to the extract loaded on the beads, FT to the flow-through fraction, and E to the elution fraction. This experiment was repeated three times.
