Poly(A) polymerase is required for RyhB sRNA stability and function in Escherichia coli

3′ETS^LeuZ drives RyhB decay in the absence of poly(A) polymerase. (A) Northern blot analysis to determine the steady-state levels of 3′ETS^LeuZ and RyhB in a wild-type (WT [fur⁺]; NRD1138) and the derived ΔpcnB strain (NRD1198) under RyhB inducing and noninducing conditions. Experiments were performed as described in the legend of Figure 5B,C. The primary glyW-cystT-leuZ transcript and the derived processing products that were detected in the northern blot are shown to the right of blot image. (B) Schematic showing complementarity (highlighted in gray) between RyhB and its target 3′ETS^LeuZ, and the specific mutations (in orange) introduced in 3′ETS^LeuZ to create a LeuZ variant (3′ETS^{LeuZ mut}) unable to pair with RyhB sRNA. (C) Northern blot analysis to determine steady-state levels of 3′ETS^LeuZ in wild-type and derived isogenic pcnB and 3′ETS^leuZ mutant strains under RyhB inducing and noninducing conditions, as described above. Northern blots showing transcript steady-state levels of 3′ETS^LeuZ and RyhB in a WT (fur⁺; NRD1138) and derived isogenic mutants 3′ETS^{leuZ WT} (fur⁺ leuZ-kan; NRD1579), 3′ETS^{leuZ mut} (fur⁺ 3′ETS^{leuZ mut}-kan; NRD1581), ΔpcnB (fur⁺ ΔpcnB; NRD1198), ΔpcnB 3′ETS^{leuZ WT} (fur⁺ ΔpcnB leuZ-kan; NRD1585), ΔpcnB 3′ETS^{leuZ mut} (fur⁺ ΔpcnB 3′ETS^{leuZ mut}-kan; NRD1587). ssrA was used as the loading control. Experiment was performed in triplicate and representative blots are shown. LeuZ term probe (Supplemental Table S2) was used to determine LeuZ and 3′ETS^LeuZ levels. (D,E) Determination of RyhB intrinsic stability in a strain encoding the wild-type 3′ETS^LeuZ (3′ETS^{leuZ WT}; NRD1579) or mutant 3′ETS^LeuZ (3′ETS^{leuZ mut}; NRD1585) and derived ΔpcnB strains, NRD1581 and NRD1587, respectively. RNA stability time-course experiments were performed as described in the legend of Figure 4. Representative northern blots are shown in C. (D) RyhB decay curves were generated as described in Figure 4 and corresponding half-life measurements are listed in Table 1. RyhB decay curves of WT (NRD1138) and its derived isogenic ΔpcnB strain (NRD1198) are included in D as a reference. Points and error bars in the curves represent the means and the standard errors (SEM) of at least three independent experiments. Probes used are listed in Supplemental Table S2.