Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts

Abstract

Escherichia coli 6S RNA represents a non-coding RNA (ncRNA), which, based on the conserved secondary structure and previous functional studies, had been suggested to interfere with transcription. Selective inhibition of sigma-70 holoenzymes, preferentially at extended −10 promoters, but not stationary-phase-specific transcription was described, suggesting a direct role of 6S RNA in the transition from exponential to stationary phase. To elucidate the underlying mechanism, we have analysed 6S RNA interactions with different forms of RNA polymerase by gel retardation and crosslinking. Preferred binding of 6S RNA to Eσ70 was confirmed, however weaker binding to Eσ38 was also observed. The crosslinking analysis revealed direct contact between a central 6S RNA sequence element and the β/β′ and σ subunits. Promoter complex formation and in vitro transcription analysis with exponential- and stationary-phase-specific promoters and the corresponding holoenzymes demonstrated that 6S RNA interferes with transcription initiation but does not generally distinguish between exponential- and stationary-phase-specific promoters. Moreover, we show for the first time that 6S RNA acts as a template for the transcription of defined RNA molecules in the absence of DNA. In conclusion, this study reveals new aspects of 6S RNA function.

Legionella pneumophila 6S RNA optimizes intracellular multiplication

“…In E. coli, genes known to be positively regulated by 6S RNA are σ S -dependent (15). The exact mechanism is currently the subject of debate and is presumably a result of the sequestration of Eσ 70 that allows the remaining core polymerase to bind to other σ factors (12,28). Comparison of genes regulated by σ S (6) and genes regulated by 6S RNA in Legionella reveals 17 genes (out of 749 genes affected by rpoS deletion or ssrS deletion) that are significantly affected in both mutants (Fig.…”

In vivo and in vitro analysis of 6S RNA-templated short transcripts inBacillus subtilis

“…16 cDNA library construction and 6S RNA reads. We have addressed the question whether B. subtilis 6S-1 and 6S-2 RNAs serve as templates for the synthesis of short pRNA transcripts, which have previously been identified in E. coli and Helicobacter pylori, 12,13,17 and more recently also in B. subtilis, but with low read coverage (ref. 18; see Discussion).…”

“…11 This highly conserved secondary structure is essential for the ability of 6S RNA to form stable complexes with σ 70 RNAP and serves as a template for the transcription of short "product RNAs" (pRNAs, 14 to 24 nucleotides) under conditions of nutrient resupply during outgrowth from stationary phase. 12,13 Synthesis of these short transcripts was reported to lead to dissociation of 6S RNA:RNAP complexes and 6S RNA degradation in the E. coli system. 14 In contrast to E. coli which harbors only a single 6S RNA gene, the B. subtilis genome By differential high-throughput RNA sequencing (dRNA-seq) we have identified "product RNAs" (pRNAs) as short as [8][9][10][11][12] nucleotides that are synthesized by Bacillus subtilis RNA polymerase (RNAp) in vivo using the regulatory 6S-1 RNA as template.…”

A new affinity approach to isolate Escherichia coli 6S RNA with histidine‐chromatography

“…Along the central bulge and through the continuous stem sequences there are mostly adenines (A) and guanines (G) (Barrick et al, 2005), which were described to interact preferably with histidine (Hoffman et al, 2004). Interestingly, in newly described aspects on the function of 6S RNA, A and G were also identified as specific nucleotides involved in close contact with RNA polymerase (Gildehaus et al, 2007).…”

“…Over the last years, new insights triggered a better understanding of the molecular mechanism of 6S RNA (Gildehaus et al, 2007;Karen, 2007), while the techniques employed in the isolation of this RNA species were still plasmid design and enzymatic purification. These techniques involve time-consuming and expensive procedures.…”