Properties of heterogeneous transcripts. (A) The effect of hfq backgrounds on the expression of heterogeneous transcripts. TM542 (ΔsgrS) and TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A600 = 0.6, 0.1% αMG was added and incubation was continued for 10 min, and then 0.02% arabinose was added and incubation was continued for 5 min. Total RNAs were prepared, and 10 or 0.25 µg of RNA samples was subjected to Northern blotting using the SgrS-S probe and tmRNA probe, respectively. Arrowheads represent the full-length transcripts. (B) An examination of Hfq binding of heterogeneous transcripts. TM803 (ΔsgrS hfq-Flag) cells harboring pSgrS-S-LS2 were grown in LB medium. At A600 = 0.6, 0.1% αMG was added and incubation was continued for 10 min, and then 0.02% arabinose was added and incubation was continued for 5 min. Crude extract was prepared and subjected to the pull-down assay using anti-Flag agarose as described in Materials and Methods. (C,D) The effect of extension of the terminator stem of sgrS-S on SgrS function in the presence (D) and absence (C) of glucose-phosphate stress. TM542 (ΔsgrS) cells harboring indicated plasmids were grown in LB medium. To prepare total RNAs from cells without the stress, 0.02% arabinose was added at A600 = 0.6 and incubation was continued for 5 min (C). To prepare total RNAs from cells with the stress, the culture of A600 = 0.6 was exposed to 0.1% αMG for 10 min, and then 0.02% arabinose for 5 min (D). Ten micrograms of RNA samples was subjected to Northern blotting using the ptsG probe.
