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

Length of pRNA_6S-2 affects the release of σ^A-RNAP from complexes with 6S-2 RNA. (A) 1.67 µM 6S-2 RNA were subjected to the folding and annealing procedure, either alone (lanes 1,2,5,8,9,12,15,16,19) or in the presence of 16.7 µM pRNA_6S-2 15-mer (lanes 3,4,6,7) or pRNA_6S-2 16-mer (lanes 10,11,13,14) or pRNA_6S-2 20-mer (lanes 17,18,20,21) in 6 µL 1× TE buffer. In lanes 1–5, 8–12, and 15–19 the 6S-2 RNA was 5′-[³²P]-end-labeled, whereas pRNAs were 5′-[³²P]-end-labeled in lanes 6, 7, 13, 14, 20, and 21. After annealing, 2 µL 5× activity buffer and 1.5 µL ddH₂O were added, and samples were kept at 37°C. Then, 0.5 µL RNAP holoenzyme (8 µg/µL) was added (lanes 2,3,7,9,10,14,16,17,21) or 0.5 µL RNAP storage buffer instead, and samples were incubated for 30 min at 37°C, followed by gel loading (f.c. σ^A-RNAP: 1 µM, f.c. 6S RNA: 1 µM, f.c. pRNA: 10 µM) and 15% nondenaturing PAGE analysis. (B) As lanes 1–4, 8–11, and 15–18 in panel A but analyzed by 7.5% nondenaturing PAGE for improved gel resolution.