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. 2019 Jul 22;47(16):8821–8837. doi: 10.1093/nar/gkz616

Figure 3.

Figure 3.

The active site of PNPase is required for decay of mRNA-derived fragments and for stabilization of sRNAs, but not for RNA binding. (A) Steady-state expression of RNA fragments in a WT (KR10000) strain containing the vector control (pTC396; WT vect) or in a Δpnp strain (NRD999; Δpnp –) containing pPNP expressing the PNPase-3x FLAG wild-type (pTC352; Δpnp WT), a derived S438A catalytic mutant (pTC354; Δpnp mut), or the vector control (pTC396; Δpnp vect). Cultures were subjected to dipyridyl treatment for 15 min immediately prior to collection of samples at OD600 of 1.0. Samples were prepared for PNPase-3xFLAG pulldowns then total RNA was isolated from a small fraction of each sample as input RNA and probed the presence of the indicated RNA fragments. SsrA served as a loading control, with 4 μg total RNA loaded per lane. (B) Immunoprecipitations were subsequently performed using anti-FLAG antibody. RNA isolated from PNPase-3xFLAG pulldown fractions was probed for the presence of RNA fragments. Each lane contains RNA collected from an equal number of cells. (C, D) Stability curves of CyaR (C) and RyhB (D) with a pnp deletion strain or derived strains harboring an empty Plac based expression vector (pTC396; vect), a plasmid expressing PNPase (pTC352; WT), or a plasmid expressing the PNPase S438A mutant (pTC354; mut). Strains were inoculated in culture media containing 100 μM IPTG to induce expression of PNPase. Expression of each sRNA was induced in exponential phase cultures for 15 min by addition of dipyridyl. Total RNA was collected at 0, 1, 2, 4 and 6 min after addition of rifampicin to inhibit further transcription, and sRNA levels were assessed by northern blot. Lines indicate best-fit exponential decay curves of three replicates, and error bars indicate the standard error of each time point. SsrA served as a loading control. For C and D, the Δrph Δpnp strain TC292 carrying cyaR under the control of the ryhB promoter and the rph-1 Δpnp strain NRD1139 carrying ryhB under its native promoter were used. Expanded views and representative northern blots are shown in Supplementary Figure S3.