A subset of Hfq-dependent sRNAs are unstable in the absence of poly(A) polymerase. RNA stability time-course experiments to determine the intrinsic stabilities of GcvB (A), MicA (B), CyaR (C), ChiX (D), and MgrR (E) sRNAs. (A,B) Strain TC279 (WT; fur+), which has MicA under the control of the ryhB promoter, and an isogenic ΔpcnB strain (ΔpcnB, DS120) were grown to exponential phase. Dipyridyl was added to each culture for 15 min to induce MicA expression, a sample was taken, rifampicin was added to block transcription, and additional samples were taken 1, 2, 4, and 6 min after rifampicin addition. RNA was extracted and northern blot analysis was performed probing for MicA or GcvB as described in Materials and Methods. To determine intrinsic stabilities of CyaR (C), ChiX (D), and MgrR (E), the wild-type strain (NRD1138) and its derived pcnB mutant (NRD1198) were grown to exponential phase, CyaR was induced from its native promoter by cAMP addition, a sample was taken after 15 min of induction, rifampicin was then added, and additional samples were taken 1, 2, 4, and 6 min after transcription inhibition. Northern blot analysis was performed using RNA extracted from these samples probing for CyaR, ChiX, and MgrR. Representative northern blots for each sRNA are shown in Supplemental Figure S1 (Supplemental Information). For the decay curves, sRNA signal intensities from the northern blots were quantified and normalized to their corresponding loading controls (ssrA or 5S). Points and error bars in the curves represent the means and the standard errors (SEM) of at least three independent experiments.
