Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs—commonalities and differences

(A) 6S-2 RNA-templated pRNA synthesis by B. subtilis σ^A-RNAP in vitro results in 6S-2 RNA:pRNA hybrids with reduced gel mobility and leads to a reduction in the amount of σ^A-RNAP:6S-2 RNA complexes. 2.5 µM 6S-2 RNA including trace amounts of 5′-[³²P]-end-labeled 6S-2 RNA (∼10,000 Cherenkov c.p.m. per gel lane) were subjected to the folding and annealing procedure in a volume of 4 µL (see Materials and Methods); then 2 µL 5× activity buffer, 1.06 µL σ^A-RNAP holoenzyme (8 µg/µL) were added (except for lane 1), and samples were incubated for 30 min at 37°C, followed by addition of 2 µL NTP solution (lanes 3–7, f.c. 200 µM each NTP) or 2 µL ddH₂O instead (lane 2, no NTPs) (final volume 10 µL; f.c. RNAP: 2 µM; f.c. 6S-2 RNA: 1 µM). After transcription at 37°C for the time period indicated above lanes 3–7, samples were analyzed by 7.5% nondenaturing PAGE. Note that the reaction was complete after 1 min. (B) 2.5 µM partially labeled 6S-2 RNA (lanes 8–12), or 2.5 µM unlabeled 6S-2 RNA together with 25 µM partially labeled pRNA_6S-2 15-mer (lane 13) were subjected to the folding and annealing procedure in a volume of 4 µL (see Materials and Methods); then, 1 µL of heparin solution (400 ng/µL) and 2 µL 5× activity buffer were added, and samples were kept at 37°C; then 1.06 µL RNAP holoenzyme (8 µg/µL) were added to samples in lanes 9–11, whereas ddH₂O was added to the samples of lanes 8, 12, and 13. Samples were incubated for 30 min at 37°C, followed by addition of 2 µL nucleotide solution (all four NTPs) to the sample of lane 11, or 2 µL of ddH₂O to the samples of lanes 8,9,12,13 (final volume 10 µL; f.c. RNAP: 2 µM; f.c. 6S-2 RNA: 1 µM; f.c. NTPs: 200 µM each). After incubation of all samples for 170 min at 37°C, 2 μL NTP solution was also added to the sample of lane 10, followed by further incubation at 37°C for 10 min, after which all samples were loaded onto a 7.5% nondenaturing PAA gel.