S-1 pRNA 9-mers are a prominent length species during outgrowth of Bacillus subtilis cells from extended stationary phase.

Bacterial RNA polymerases (RNAP) utilize 6S RNAs as templates to synthesize ultrashort transcripts (up to ~ 14 nt), termed product RNAs (pRNAs), that play a key role in reversing the blockage of RNAP by 6S RNA. Here we resolved the pRNA length profile of 6 S-1 RNA from Bacillus subtilis, a major model system for the study of 6S RNA biology, during outgrowth of cells from extended stationary phase. 9-mers were found to be a particularly abundant pRNA length species, followed by 8/10/11-mers and 13/14-mers. Consistent with in vitro data from the Escherichia coli system, these findings support the mechanistic model according to which the housekeeping sigma factor (σ⁷⁰ or σ^A) dissociates from 6S RNA:RNAP complexes upon synthesis of pRNA 9-mers, followed by final dissociation of 6S RNA and RNAP upon synthesis of longer pRNAs (13/14-mers). Methodologically, the identification of such ultrashort RNAs in total cellular extracts by RNA-Seq is inefficient with standard protocols using adapter ligation to RNA 3'-ends for reverse transcription and PCR-based cDNA sequencing. Here, we demonstrate that ultrashort RNAs can instead be incorporated into RNA-Seq libraries by polyA, polyC and potentially also polyU tailing of their 3'-ends. At positions where a non-tailing nucleotide is followed by one or more tailing nucleotides, an algorithm that integrates RNA-Seq results from at least two different 3'-end tailings allows one to approximate the fraction of read counts at such ambiguous positions. Finally, methodological biases and potential applications of our approach to other short RNAs are discussed.