Northern blot analysis of 6S-1 RNA processing and decay in the B. subtilis wt (W168) strain and RNase knockout derivative strains. (A) Total RNAs isolated from stationary (stat.) phase cells of the wt (lane 1), ΔrnjA (ΔRNase J1, lane 3), Δrph (ΔRNase PH, lane 4) strain, and the quadruple mutant Δ4exo (lane 5) with knockouts of rph, rnr (RNase R), yhaM (YhaM), and pnp (polynucleotide phosphorylase, PNPase). In the case of Δrny (ΔRNase Y, lane 2) bacteria, RNA was not isolated at the final stationary phase, but at an intermediate stagnation phase at 0.4–0.8 OD600 (Supplemental Fig. S1), termed stat1 phase. RNAs were separated by 10% denaturing PAGE. Lane 6: a 197 nt T7 transcript of 6S-1 RNA loaded as size marker (M). (B) Northern blot analysis after extended electrophoresis to resolve the region between precursor and mature 6S-1 RNA. Lanes 1–3: total RNA isolated from ΔrnjA bacteria in stationary (stat.) phase (lane 1) or after 3 min (lane 2) and 30 min (lane 3) of outgrowth (1:5 dilution of stationary cells in fresh LB medium); lanes 4,5: total RNA isolated from stationary phase cells of the wt and Δrph strains; lanes 6,7: T7 transcripts of mature (197 nt) and precursor (208 nt) 6S-1 RNA used as size markers (for details, see Supplemental Methods). The shown blots are representative of 10 (panel A) or five (panel B) individual blots using two to four independent RNA preparations. (C) 6S-1 RNA northern blot analysis (12% denaturing PAGE) using total RNA from Δrny bacteria in the final stationary (stat2) phase (lane 2) or 30 min after induction of outgrowth (lane 6). RNA preparations from wt and Δ4exo strains harvested at the corresponding growth stages were loaded for comparison (lanes 1,5,7). The 6S-2 RNA marker transcripts (lanes 4,9) were loaded to control for probe specificity. The shown blots are representative of at least three individual blots performed with independent RNA preparations. For the specificity of probe 6S-1_mature, see Supplemental Figure S2A. 5S rRNA was used as loading control in panels A and C. 6S-1 RNA and 5S rRNA were probed simultaneously (panel A) or successively (panel C) using full-length probes complementary to 6S-1 RNA and 5S rRNA (Table 1; Fig. 1); we confirmed in preceding northern blot experiments that the 5S RNA probe only gives rise to a single 5S rRNA-specific signal (indicated at the left margin) under the applied conditions.
