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

Abstract

(2011) In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillussubtilis, RNA Biology, 8:5,[839][840][841][842][843][844][845][846][847][848][849]

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

“…The RNA was observed to reach highest steadystate levels between early and mid-exponential phase and to decrease toward stationary phase Barrick et al 2005;Beckmann et al 2011). Deviating from these observations, B. subtilis 6S-2 RNA levels were also reported to remain fairly constant in exponential and stationary phase (Trotochaud and Wassarman 2005;Cavanagh et al 2012).…”

“…We have demonstrated in vitro that the σ A -RNAP holoenzyme is able to synthesize pRNAs on 6S-2 RNA as a template (Beckmann et al 2011). So far, deep sequencing analyses have not provided evidence for the synthesis of 6S-2 RNA-derived pRNAs in vivo (Irnov et al 2010;Beckmann et al 2011).…”

“…Both RNAs were shown to coimmunoprecipitate with the σ A housekeeping RNAP holoenzyme using antibodies against σ A or the core subunit α (Trotochaud and Wassarman 2005). Recent investigations provided evidence that 6S-1 RNA displays all of the hallmarks of a canonical 6S RNA: (1) It reaches its highest steady-state levels during late exponential/early stationary phase (Barrick et al 2005;Beckmann et al 2011); (2) it serves as an RNA template for massive pRNA transcription, particularly during outgrowth from stationary phase upon resupply of nutrients, as inferred from deep sequencing (dRNA-seq) (Beckmann et al 2011) and Northern blot analyses (Beckmann et al 2011;Cavanagh et al 2012); and (3) longer pRNAs (12-to 14-mers), whose fraction increases during outgrowth from stationary phase (Beckmann et al 2011), form a stable duplex with 6S-1 RNA and induce a structural rearrangement in 6S-1 RNA. The rearranged 6S-1 RNA:pRNA complexes lose their affinity for σ A -RNAP to liberate σ A -RNAP for transcription at DNA promoters during the newly initiated exponential phase.…”

“…Depending on the particular expression profile of a 6S RNA, the RNA sequesters a substantial fraction of the housekeeping RNAP holoenzyme, which then becomes unavailable for transcriptional activity at DNA promoters (Wassarman 2007). This block can be relieved when RNAP synthesizes so-called product RNAs (pRNAs) of a certain length utilizing 6S RNA itself as a transcription template, which then triggers the release of RNAP (Wassarman and Saecker 2006;Gildehaus et al 2007;Beckmann et al 2011).…”

E. coli 6S RNA release from RNA polymerase requires σ70 ejection by scrunching and is orchestrated by a conserved RNA hairpin

“…In the absence of heparin, pRNA sizes increase (Supplemental Fig. S2) but appear to have limited in vivo significance; as pRNAs of length z10-14 nt are known to be produced within 15-30 sec of rapid outgrowth in E. coli (Wurm et al 2010) and are very similar in size to the pRNAs observed in H. pylori (12 nt long) ) and B. subtilis (8-12 nt long) (Beckmann et al 2011). Longer RNAs complementary to a pRNA probe have been detected by Northern blot from E. coli, but they start to accumulate only after 1-2 min of outgrowth (Wurm et al 2010).…”