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

A pRNA_6S-2 15-mer is less effective than a pRNA_6S-1 14-mer in promoting release of the cognate 6S RNA from σ^A-RNAP. (A) 2.5 μM 6S-1 RNA including trace amounts of 5′-[³²P]-end-labeled 6S-1 RNA were subjected to the folding and annealing procedure, either alone (lanes 1 and 2) or in the presence of 25 µM pRNA_6S-1 14-mer (lanes 3 and 4) or 25 µM pRNA_6S-2 15-mer as a noncomplementary control (lanes 5 and 6) in 4 µL 1×TE buffer. Then, 2 µL 5× activity buffer and 3.5 µL ddH₂O were added, and samples were kept at 37°C before adding 0.5 µL RNAP holoenzyme (8 µg/µL) (lanes 2,3,5) or 0.5 µL RNAP storage buffer instead (lanes 1,4,6), followed by incubation for 30 min at 37°C (f.c. σ^A-RNAP: 1 µM, f.c. 6S RNA: 1 µM, f.c. pRNA: 10 µM), gel loading, and 7.5% nondenaturing PAGE. (B) The analogous experiment with 6S-2 RNA.