Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides.

Transcription of rRNAs in Escherichia coli is directed from seven redundant rRNA operons, which are mainly regulated by their P1 promoters. Here we demonstrate by in vivo measurements that the amounts of individual rRNAs transcribed from the different operons under normal growth vary noticeably although the structures of all the P1 promoters are very similar. Moreover, we show that starvation for amino acids does not affect the seven P1 promoters in the same way. Notably, reduction of transcription from rrnD P1 was significantly lower compared to the other P1 promoters. The presence of DksA was shown to be crucial for the ppGpp-dependent downregulation of all P1 promoters. Because rrnD P1 is the only rrn promoter starting with GTP instead of ATP, we performed studies with a mutant rrnD promoter, where the initiating G+1 is replaced by A+1. These analyses demonstrated that the ppGpp sensitivity of rrn P1 promoters depends on the nature and concentration of initiating nucleoside triphosphates (iNTPs). Our results support the notion that the seven rRNA operons are differentially regulated and underline the importance of a concerted activity between ppGpp, DksA and an adequate concentration of the respective iNTP.

Rare codons play a positive role in the expression of the stationary phase sigma factor RpoS (σ(S)) in Escherichia coli.

Rare codons can influence the stability of messenger RNAs, promote regular spacing of ribosomes on a transcript, or modulate stability and proper folding of nascent proteins. The mRNA specifying the stationary phase master regulator RpoS, which belongs to the RpoD family of sigma factors, contains a high number of rare codons, including many codons at positions corresponding to more frequent codons encoding the same amino acids in the homologous RpoD sequence. Substituting these rare codons in rpoS by the more frequent synonymous rpoD codons resulted in decreased transcript and protein levels compared to the natural rare-codon wildtype version of rpoS. The frequent-codon mutant rpoS transcript exhibited faster turnover than the rare-codon wildtype mRNA. Studies with endoribonuclease-deficient strains revealed RNase E to be crucial for this accelerated mRNA degradation. Thus, in the case of RpoS expression, "less is obviously more", as our data suggest a model, in which slowing down translational speed by ribosomal pausing at many rare codons along a transcript could reduce ribosome spacing and thereby protect the transcript against ribonucleolytic attack by RNase E. Such a mechanism may be especially important for translationally controlled genes like rpoS where the formation of secondary structure in the translational initiation region competes with (therefore relatively inefficient) ribosome loading. Moreover, strong codon differences in genes encoding isoenzymes expressed in exponential and stationary phase suggest that transcript protection by repetitive ribosome pausing at multiple rare codons in stationary phase-expressed transcripts may be a general principle to save resources under nutrient-limited conditions.

The influence of Hfq and ribonucleases on the stability of the small non-coding RNA OxyS and its target rpoS in E. coli is growth phase dependent.

OxyS is one of at least three small non-coding RNAs, which affect rpoS expression. It is induced under oxidative stress and reduces the levels of the stationary phase sigma factor RpoS. We analyzed the turn-over of OxyS and rpoS mRNA in early exponential and in stationary growth phase in different E. coli strains to learn more about the mechanisms of processing and about a possible impact of processing on growth-dependent regulation. We could not attribute a major role of RNase E, RNase III, PNPase or RNase II on OxyS turn-over in exponential growth phase. Only the simultaneous lack of RNase E, PNPase and RNase II activity resulted in some stabilization of OxyS in exponential growth phase, implying the action of multiple ribonucleases on OxyS turn-over. A major role of RNase E on OxyS stability was observed in stationary phase and was dependent on the presence of the RNA binding protein Hfq and of DsrA, one of the other small RNAs binding to rpoS mRNA. Our data also confirm a role of RNase III in rpoS turn-over, however, only in exponential growth phase.We conclude that OxyS and rpoS mRNA processing is influenced by different RNases and additional factors like Hfq and DsrA and that the impact of these factors is strongly dependent on growth phase.