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

Abstract

Background: 6S 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 sigma(70) (Esigma(70)) it is believed that 6S RNA supports adjustment to stationary phase transcription. Previous studies have also suggested that inhibition is specific for sigma(70)-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 (sigma(38)-dependent) promoters are inhibited.

Results: To 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 > or = 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.

Conclusions: The analysis demonstrated that the effect of 6S RNA on transcription is not strictly confined to sigma(70)-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.

Figure 1

Primer extension analysis of selected genes. Primer extension results are exemplified for selected promoters. Total RNA was isolated from MM139 or MC4100 cells. Different growth phases for RNA isolation are indicated by A: early log (A₆₀₀ = 0.4), B: late log (A₆₀₀ = 1.2), C: early stationary (A₆₀₀ = 2.7), D: stationary phase (A₆₀₀ = 3.3). The presence of functional 6S RNA is indicated by -- or +, respectively. The predominant sigma factor specificity is indicated in brackets below the respective genes. Note that rpoD P3 is a minor promoter, which we have assigned as σ⁷⁰-dependent according to its consensus sequence. Transcription of this promoter is also affected by the heat shock-specific sigma factor σ³²[48].

Figure 2

Volcano plot of global gene expression differences. The plot indicates global gene expression data (●) of the 6S deficient strain MM139 and WT in the early stationary phase. Expression of ~50 genes encoding ribosomal proteins and proteins involved in transcription (rpoA, rpoB, rpoC) and translation elongation (tufA, fusA) showed almost uniformly slightly decreased and up to 2-fold lower expression levels (●).

Figure 3

Comparison between microarray and primer extension results. a) Results from primer extension analyses of selected genes are presented. RNA samples were isolated at exponential or early stationary phase (exp. or stat., respectively). In b) quantitative evaluation (ratio of relative transcripts from ssrS^-/ssrS⁺ from two to four independent experiments) of the primer extension results for selected genes and the corresponding results from the microarray analysis for the early stationary phase are shown. * hisL, the leader region of the his operon, has not been found differentially expressed by the microarrays but is 6S RNA sensitive according to primer extension.

Figure 4

6S RNA affects ribosomal RNA transcription at stationary growth. a) Primer extension analysis of bulk rRNA transcription from P1 and P2 promoters. Two independent RNA samples were analyzed from ssrS^- (-) and ssrS⁺ (+) cells grown exponentially (exp.) or stationary (stat.). cDNA products originating from P1 and P2 promoters are indicated. Multiple bands are resolved due to sequence heterogeneities of the rRNA leader regions from the seven different rRNA operons. The constitutively expressed rhoL transcript served as an internal standard for quantification. Lanes 1 and 5: RNA from ssrS^- cells at exponential growth, lanes 2 and 6: RNA from ssrS⁺ cells at exponential growth, lanes 3 and 7: RNA from ssrS^- cells at stationary growth, lanes 4 and 8: RNA from ssrS⁺ cells at stationary growth. b) Quantitative evaluation of P2 transcription products at early stationary phase from RNA samples of ssrS^- (Mutant) and ssrS⁺ (wild-type) cells is shown. Error bars give the standard deviation of 4 independent experiments.

Figure 5

During stationary growth the basal ppGpp level is increased in 6S RNA deficient strains. NTPs extracted after in vivo labelling from two independent experiments were separated by thin layer chromatography. Lanes 1 and 3: extracts from ssrS^- strain, lanes 2 and 4: extracts from the wild-type MC4100, lanes 5 and 6: extract from the relA⁺ strain MG1655. The sample in lane 6 had been treated with serine hydroxamate to induce the stringent control in order to produce high levels of ppGpp and pppGpp, which served as mobility markers.