FIGURE 5.

RapZ overproduction may compensate for loss of its RNA-binding activity in vivo. Complementation experiment addressing the activities of plasmid encoded RapZ_quad and RapZ_1–279 variants in vivo. Plasmids triggering either low (lanes 4–6: pBGG61, pYG30, pSD128) or high (lanes 8–10: pBGG164, pYG29, pSD135) expression levels of the rapZ variants from the P_Ara or the P_tac promoter, respectively, were tested in strain Z37, which lacks endogenous rapZ. Empty plasmids pBAD33 and pBGG237 provided the vector controls, respectively (VC; lanes 3 and 7). In lane 1, wild type strain R1279 was analyzed for comparison. For induction of the P_Ara promoter, 0.2% arabinose was added. Total RNA and protein were isolated from exponentially growing cells and analyzed by northern and western blotting, respectively, for detection of GlmZ, 5S rRNA (loading control), GlmS and protein S2 (loading control). In the bottom panel, total protein extracts were separated on a SDS-PAA gel and stained with Coomassie blue to verify overproduction of the RapZ variants from the P_tac constructs (indicated by arrows).