Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.

Exposure of E. coli to hydrogen peroxide induces the transcription of a small RNA denoted oxyS. The oxyS RNA is stable, abundant, and does not encode a protein. oxyS activates and represses the expression of numerous genes in E. coli, and eight targets, including genes encoding the transcriptional regulators FhlA and sigma(S), were identified. oxyS expression also leads to a reduction in spontaneous and chemically-induced mutagenesis. Our results suggest that the oxyS RNA acts as a regulator that integrates adaptation to hydrogen peroxide with other cellular stress responses and helps to protect cells against oxidative damage.

Dps is a non-specific DNA-binding protein abundant in starved Escherichia coli cells and is important for the defence against hydrogen peroxide. We found that dps mRNA levels are controlled by rpoS-encoded sigma S, the transcriptional activator OxyR and the histone-like IHF protein. In exponentially growing cells, dps is induced by treatment with hydrogen peroxide in an OxyR-dependent manner. This OxyR-dependent induction occurs only during log phase, although the OxyR protein is present in stationary phase. In the stationary phase cells, dps is expressed in a sigma S- and IHF-dependent manner. The purified OxyR and IHF proteins are also shown to bind upstream of the dps promoter. Our results suggest that the dps promoter is recognized by both sigma 70-holoenzyme and sigma S-holoenzyme, since OxyR acts through sigma 70 and the starts of the OxyR- and sigma S-dependent transcripts are identical.

The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded σ s subunit of RNA polymerase. σ s is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfqencoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.

The importance of post-transcriptional regulation by small non-coding RNAs has recently been recognized in both pro-and eukaryotes. Small RNAs (sRNAs) regulate gene expression posttranscriptionally by base pairing with the mRNA. Here we use dynamical simulations to characterize this regulation mode in comparison to transcriptional regulation mediated by protein-DNA interaction and to post-translational regulation achieved by protein-protein interaction. We show quantitatively that regulation by sRNA is advantageous when fast responses to external signals are needed, consistent with experimental data about its involvement in stress responses. Our analysis indicates that the half-life of the sRNA-mRNA complex and the ratio of their production rates determine the steady-state level of the target protein, suggesting that regulation by sRNA may provide fine-tuning of gene expression. We also describe the network of regulation by sRNA in Escherichia coli, and integrate it with the transcription regulation network, uncovering mixed regulatory circuits, such as mixed feed-forward loops. The integration of sRNAs in feedforward loops provides tight repression, guaranteed by the combination of transcriptional and post-transcriptional regulations.

The emergence of pathogenic strains of enteric bacteria and their adaptation to unique niches are associated with the acquisition of foreign DNA segments termed ‘genetic islands’. We explored these islands for the occurrence of small RNA (sRNA) encoding genes. Previous systematic screens for enteric bacteria sRNAs were mainly carried out using the laboratory strain Escherichia coli K12, leading to the discovery of ∼80 new sRNA genes. These searches were based on conservation within closely related members of enteric bacteria and thus, sRNAs, unique to pathogenic strains were excluded. Here we describe the identification and characterization of 19 novel unique sRNA genes encoded within the ‘genetic islands’ of the virulent strain Salmonella typhimurium. We show that the expression of many of the island-encoded genes is associated with stress conditions and stationary phase. Several of these sRNA genes are induced when Salmonella resides within macrophages. One sRNA, IsrJ, was further examined and found to affect the translocation efficiency of virulence-associated effector proteins into nonphagocytic cells. In addition, we report that unlike the majority of the E. coli sRNAs that are trans regulators, many of the island-encoded sRNAs affect the expression of cis-encoded genes. Our study suggests that the island encoded sRNA genes play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus controls virulence.

More than 60 small RNAs (sRNA) have been identified in E. coli. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

The importance of small, noncoding RNAs that act as regulators of transcription, of RNA modification or stability, and of mRNA translation is becoming increasingly apparent. Here we discuss current knowledge of regulatory RNA function and review how the RNAs have been identified in a variety of organisms. Many of the regulatory RNAs act through base-pairing interactions with target RNAs. The base-pairing RNAs can be grouped into two general classes: those that are encoded on the opposite strand of their target RNAs such that they contain perfect complementarity with their targets, and those that are encoded at separate locations on the chromosome and have imperfect base-pairing potential with their targets. Other regulatory RNAs act by modifying protein activity, in some cases by mimicking the structures of other RNA or DNA molecules.