Northern blot analysis of 6S-1 pRNA synthesis and decay in B. subtilis. Endogenous pRNAs (∼14 nt), synthesized from 6S-1 RNA as template, were detected in total RNA extracts (6 µg) withdrawn from wt and ΔrnjA cells 3 min after induction of outgrowth (lanes 1,2). As specificity control, total RNA from a 6S-1 RNA knockout strain (ΔbsrA, lanes 3,9) was analyzed in parallel. A chemically synthesized pRNA 14-mer (5′-GUUCGGUCAAAACU-3′) was loaded in two different amounts (0.25 and 1 ng) as length marker (lanes 4,5,10,11). To identify RNases involved in 6S-1 pRNA decay, 10 µg of total RNA from wt cells, the quadruple mutant (Δ4exo), ΔrnjA and ΔbsrA bacteria, harvested 30 min after outgrowth, were analyzed by 10% native PAGE and northern blotting (lanes 6–9). The shown blots are representative of four individual blots performed with two to three independent RNA preparations; pRNAs were detected by probe 6S-1_pRNA.
