The effect of extension of the terminator stem of sgrS-S on the generation of heterogeneous shorter transcripts. (A) DNA sequences of sgrS-S and its variants. The inverted repeat sequences of the terminator are indicated by horizontal arrows. The inserted sequences to stabilize the terminator stem are shown as bold letters in sgrS-S-LS1 and sgrS-S-LS2. The sequences derived from the rrnBT and rplLT are shown as bold letters in sgrS-S-LS3 and sgrS-S-LS4, respectively. Nucleotides are numbered from the site corresponding to the 5′ end of sgrS-S. (B) The predicted secondary structures and the thermodynamic stabilities (ΔG, kcal/mol) of terminator RNA hairpins without a poly(U) sequence were determined according to the Mfold program (Zuker 2003). (C) The effect of extension of the terminator stem of sgrS-S on the generation of shorter transcripts. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A600 = 0.6, 0.2% arabinose was added and incubation was continued for 10 min. Total RNAs were prepared, and 20 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. (D) The effect of extension of the terminator stem of sgrS-S on the generation of shorter transcripts under glucose-phosphate stress. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium. At A600 = 0.6, 0.01% αMG was added and incubation was continued for 10 min. Then, 0.2% arabinose was added and incubation was continued for 10 min. Total RNAs were prepared, and 20 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. (E) The effect of replacement of the terminator hairpin of sgrS-S with those derived from rrnBT and rplLT on the generation of shorter transcripts. TM772 (ΔsgrS Δhfq) cells harboring indicated plasmids were grown in LB medium and treated as described in D. Total RNAs were prepared, and 20 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.
