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

The extent of inhibition of pRNA de novo transcription by preformed 6S RNA:pRNA complexes depends on the pRNA length. (A) 6S-2 RNA (2 µM) was subjected to the folding and annealing procedure in 5 µL of 1× TE buffer, either alone (lane 4) or in the presence of 40 µM pRNA_6S-2 20-mer (lane 5), 16-mer (lane 6), 15-mer (lane 7), 14-mer (lane 8), 13-mer (lane 9), 12-mer (lane 10), or pRNA_6S-1 14-mer as control (lane 11, noncomplementary pRNA). After annealing, 0.5 µL water, 2 µL 5× activity buffer, and 0.5 µL σ^A-RNAP (8 µg/µL) were added, and reactions were incubated for 30 min at 37°C followed by addition of 2 µL of NTP mix (f.c. 200 µM each NTP, also containing α-[³²P]-ATP) and further incubation for 30 min at 37°C. As size markers, 5′-[³²P]-end-labeled pRNA_6S-2 20-mer (lane 2) and 15-mer (lane 3) were used. (B) 6S-1 RNA (2 µM) was subjected to the folding and annealing procedure in 5 µL of 1× TE buffer, either alone (lane 14) or in the presence of 40 µM pRNA_6S-1 14-mer (lane 15), 13-mer (lane 16), 12-mer (lane 17), or pRNA_6S-2 15-mer as control (lane 18, noncomplementary pRNA). In vitro transcription by σ^A-RNAP was performed as in panel A, except that the NTP mix contained α-[³²P]-UTP instead of α-[³²P]-ATP. 5′-[³²P]-end-labeled pRNA_6S-1 14-mer (lane 13) and 8-mer (lane 19) were loaded as size markers. Lanes 1 and 12 are negative transcription controls where 6S RNA was omitted. Transcription products were analyzed by 25% denaturing PAGE.