FIG. 4.

rpoS mRNA half-life determination when it is paired with DsrA or RprA. CM1000 (ΔdsrA rpoS⁺)/pBAD-dsrA and CM1001 (ΔdsrA rpoS*)/pBAD-dsrA* (A) and JNB001 (ΔdsrA rprA::kan rpoS⁺) and JNB002 (ΔdsrA rprA::kan rpoS*) with pBAD-rprA or pBAD-rprA* (B) were grown in LB Ap at 30°C to mid-exponential phase, and sRNA expression was induced with 0.02% arabinose for 15 min. RNA samples were collected by the hot phenol method at the indicated times after the addition of 250 μg/ml rifampin (rif). rpoS mRNA levels were analyzed by Northern blotting. The experiment whose results are shown in panel A was performed three times, resulting in half-lives measured for rpoS and rpoS* of 2 to 3 min in each case. The experiment whose results are shown in panel B was performed four times, with some variability in half-lives being measured for rpoS and rpoS*. In each experiment, the mRNA half-lives of the mismatched pairs (rpoS⁺-rprA* and rpoS*-rprA⁺) are shorter than that of the optimally paired sets. However, the mRNA half-lives in the rpoS⁺-rprA⁺ and rpoS*-rprA* strains varied from 4 to 15 min, and the mRNA half-lives in the mismatched pairs varied from 1 to 10 min. (C and D) Graphical representation of rpoS mRNA decay.