Bacillus subtilis 6S-2 RNA serves as a template for short transcripts in vivo.

The global transcriptional regulator 6S RNA is abundant in a broad range of bacteria. The RNA competes with DNA promoters for binding to the housekeeping RNA polymerase (RNAP) holoenzyme. When bound to RNAP, 6S RNA serves as a transcription template for RNAP in an RNA-dependent RNA polymerization reaction. The resulting short RNA transcripts (so-called product RNAs = pRNAs) can induce a stable structural rearrangement of 6S RNA when reaching a certain length. This rearrangement leads to the release of RNAP and thus the recovery of transcription at DNA promoters. While most bacteria express a single 6S RNA, some harbor a second 6S RNA homolog (termed 6S-2 RNA in Bacillus subtilis). Bacillus subtilis 6S-2 RNA was recently shown to exhibit essentially all hallmark features of a bona fide 6S RNA in vitro, but evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo has been lacking so far. This raised the question of whether the block of RNAP by 6S-2 RNA might be lifted by a mechanism other than pRNA synthesis. However, here we demonstrate that 6S-2 RNA is able to serve as a template for pRNA synthesis in vivo. We verify this finding by using three independent approaches including a novel primer extension assay. Thus, we demonstrate the first example of an organism that expresses two distinct 6S RNAs that both exhibit all mechanistic features defined for this type of regulatory RNA.