Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising reduction in the expression of the translation machinery

Abstract

Background6S RNA from E. coli is known to bind to RNA polymerase interfering with transcription initiation. Because 6S RNA concentrations are maximal at stationary phase and binding occurs preferentially to the holoenzyme associated with σ70 (Eσ70) it is believed that 6S RNA supports adjustment to stationary phase transcription. Previous studies have also suggested that inhibition is specific for σ70-dependent promoters characterized by a weak -35 recognition motif or extended -10 promoters. There are many exceptions to this precept, showing that other types of promoters, including stationary phase-specific (σ38-dependent) promoters are inhibited.ResultsTo solve this apparent ambiguity and to better understand the role of 6S RNA in stationary phase transition we have performed a genome-wide transcriptional analysis of wild-type and 6S RNA deficient cells growing to mid-log or early stationary phase. We found 245 genes at the exponential growth phase and 273 genes at the early stationary phase to be ≥ 1.5-fold differentially expressed. Up- and down-regulated genes include many transcriptional regulators, stress-related proteins, transporters and several enzymes involved in purine metabolism. As the most striking result during stationary phase, however, we obtained in the 6S RNA deficient strain a concerted expression reduction of genes constituting the translational apparatus. In accordance, primer extension analysis showed that transcription of ribosomal RNAs, representing the key molecules for ribosome biogenesis, is also significantly reduced under the same conditions. Consistent with this finding biochemical analysis of the 6S RNA deficient strain indicates that the lack of 6S RNA is apparently compensated by an increase of the basal ppGpp concentration, known to affect growth adaptation and ribosome biogenesis.ConclusionsThe analysis demonstrated that the effect of 6S RNA on transcription is not strictly confined to σ70-dependent promoters. Moreover, the results indicate that 6S RNA is embedded in stationary phase adaptation, which is governed by the capacity of the translational machinery.

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

“…We additionally analyzed four B. subtilis promoters (rrnO, argC, appD, cspB) and one B. subtilis phage ϕ29 promoter (C2ϕ29), which showed similar trends as the veg and rrnB P1 promoters (data not shown). Although we are aware that inhibition in vitro at a few DNA promoters does not allow conclusions about which genes are affected by 6S-1/2 RNAs in vivo on a global scale (Cavanagh et al 2008;Neusser et al 2010), our findings provide first evidence that 6S-1 and 6S-2 RNAs have comparable capacities to effectively compete with DNA promoters for binding to σ A -RNAP, in line with their very similar affinities for the enzyme (Fig. 3).…”

6S RNA – an old issue became blue-green

“…The amount of 6S RNA to reach half-maximal inhibition varied slightly for the different 6S RNAs and was also not identical for the individual promoters tested. The observed differences in the degree of inhibition for the different cyanobacterial 6S RNAs can be explained by subtle differences in the affinities of the RNAs for E. coli RNA polymerase, but may also reflect an unidentified promoter-specific component, a property which is also known for E. coli 6S RNA (Cavanagh et al, 2008;Gildehaus et al, 2007;Neußer et al, 2010). The addition of increasing concentrations of tRNA did not affect the transcription of any promoters notably, underlining the specificity of the 6S RNAs to act as transcriptional repressors.…”

“…2010). Our previous studies also revealed that there is a link between 6S RNA and the global regulator ppGpp, which affects growth rate, ribosome synthesis and the translational capacity of the cell (Cavanagh et al, 2010;Neußer et al, 2010). A particularly striking discovery was the observation that 6S RNA can serve as a template for the de novo transcription of small RNAs [de novo RNAs (dnRNAs) or product RNAs (pRNAs)] when stationary cells encounter better nutritional conditions (Gildehaus et al, 2007;Wassarman & Saecker, 2006;Wurm et al, 2010).…”

“…This leads to the inhibition of transcription for many but not all s 70 -dependent promoters, and facilitates the adaptation to stationary phase and environmental stress (Cavanagh et al, 2008;Gildehaus et al, 2007;Trotochaud & Wassarman, 2004, 2006. Additional functions for 6S RNA have been discovered by genome-wide transcriptome studies, indicating that the molecule is important for central steps in the metabolism of the cell, such as growth phase adaptation or carbon and purine metabolism (Geißen et al, 2010;Neußer et al, 3These authors contributed equally to this work. 2010).…”

Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli

“…2) raise the question of whether it is subject to 6S RNA regulation. 6S RNA-dependent phenotypes are very complex and often contextual with respect to growth conditions (Cavanagh et al, 2008;Neusser et al, 2010). To the best of our knowledge, there is no published evidence for 6S RNA regulation of LEE gene expression.…”