Processing and decay of 6S-1 and 6S-2 RNAs in Bacillus subtilis

In vitro RNase J1 cleavage of precursor 6S-1 RNA using either mono- or triphosphorylated substrates. Lanes 1,2: monophosphorylated, 5′-³²P-endlabeled pre-6S-1 RNA carrying two additional G residues at the 5′-end for efficient T7 transcription, incubated with (lane 2) or without (lane 1) RNase J1; lanes 3,4: as lanes 1,2, but using the same RNA with a triphosphorylated (γ-³²P) 5′-end; lanes 5,6,8,9: a 13 nt long synthetic RNA oligonucleotide (5′-GGAAGUUAAAUAA-3′) corresponding to the 5′-leader of the pre-6S-1 RNA analyzed in lanes 1–4, incubated with (lanes 5,9) or without nuclease P1 (lanes 6,8); lane 7: γ-³²P-GTP; lanes 10,11: as lanes 1,2, but using monophosphorylated, 5′-³²P-endlabeled pre-6S-1 RNA starting with the native 5′-terminal A residue (i.e., lacking the two additional G residues). Asterisks next to lanes 4,11 indicate additional larger cleavage products. For more details, see Materials and Methods.