Analysis of 6S-2 RNA processing and decay. (A) Northern blot detection of 6S-2 RNA and fragments thereof (12% denaturing PAGE, probe 6S-2_mature) in total RNA preparations from wt cells harvested in early (OD600 = 1.35; at 3.2 h) and late (OD600 = 3.2; at 5.2 h) exponential as well as stationary phase (OD600 = 2.4; at 27 h). Full-length antisense 6S-2 RNA (probe 6S-2_mature, dark blue line in Fig. 9) was used as the probe. M (lane 4), T7 transcript of 6S-2 RNA, 209 nt in length (for details, see Supplemental Material). 5S rRNA served as a loading control. 6S-2s stands for 6S-2shortened, a processing product somewhat shorter than full-length 6S-2 RNA. (B) Corresponding northern blot analysis of 6S-2 RNA decay in the wt and RNase knockout derivative strains (10% denaturing PAGE). Total RNA was isolated from stationary phase cells, except for Δrny stat1 representing the intermediate stagnation phase at 0.4–0.8 OD600 (Supplemental Fig. S1). (C) As in panel B, but using the 5′-end-specific probe 6S-2_5′ (sky blue line in Fig. 9). The identity of the lower signal as fragment “c” was inferred from its migration distance relative to the full-length RNA signal (gels in panels B and C were run identically) and further confirmed in Supplemental Figure S7C. The shown blots are representative of 10 (panel B) or three (panel C) individual blots using two to five independent RNA preparations. (D) Percentage of RNA-seq 5′-end reads mapping to the 6S-2 RNA locus up to nt position 60 in ΔrnjA library 2 (green bars); gray bars indicate the values for the respective library 2 of the wt strain. (E) Northern blot analysis (12% denaturing PAGE) of 6S-2 RNA using total RNA derived from stationary cells of the wt strain, the Δrny strain (stat2, terminal stationary phase), and the Δ4exo strain using probe 6S-2_mature and different RNA concentrations as indicated above the blots. M, 209 nt T7 transcript used as marker for mature 6S-2 RNA. The swung dashes next to lanes 3, 6 and 10 mark the upshifted fragment “a” in the Δ4exo lanes. (F) RNA-seq profiles showing depletion of mature 3′-end reads (position 211) and simultaneous accumulation of 3′-end reads representing 6S-2 RNAs with their 3′-end extended by 3 nt (3′-end at position 214) in the Δrph and Δ4exo strains (green and magenta bars) relative to the library 1 wt strain (gray bars). The percentages of 3′-ends at positions 211–214 in the libraries for the wt and mutant strains are indicated below the corresponding bars in the same color code.
