Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.     

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA-RNA interactions.     

Major role for mRNA binding and restructuring in sRNA recruitment by Hfq

Bacterial small RNAs (sRNAs) modulate gene expression by base-pairing with target mRNAs. Many sRNAs require the Sm-like RNA binding protein Hfq as a cofactor. Well-characterized interactions between DsrA sRNA and the rpoS mRNA leader were used to understand how Hfq stimulates sRNA pairing with target mRNAs. DsrA annealing stimulates expression of rpoS by disrupting a secondary structure in the rpoS leader, which otherwise prevents translation. Both RNAs bind Hfq with similar affinity but interact with opposite faces of the Hfq hexamer. Using mutations that block interactions between two of the three components, we demonstrate that Hfq binding to a functionally critical (AAN)(4) motif in rpoS mRNA rescues DsrA binding to a hyperstable rpoS mutant. We also show that Hfq cannot stably bridge the RNAs. Persistent ternary complexes only form when the two RNAs are complementary. Thus, Hfq mainly acts by binding and restructuring the rpoS mRNA. However, Hfq binding to DsrA is needed for maximum annealing in vitro, indicating that transient interactions with both RNAs contribute to the regulatory mechanism.     

Interactions of ribosomal protein S1 with DsrA and rpoS mRNA

Ribosomal protein S1 is shown to interact with the non-coding RNA DsrA and with rpoS mRNA. DsrA is a non-coding RNA that is important in controlling expression of the rpoS gene product in Escherichia coli. Photochemical crosslinking, quadrupole-time of flight tandem mass spectrometry, and peptide sequencing have identified an interaction between DsrA and S1 in the 30S ribosomal subunit. Purified S1 binds both DsrA (K(obs) approximately 6 x 10(6) M(-1)) and rpoS mRNA (K(obs) approximately 3 x 10(7) M(-1)). Ribonuclease probing experiments indicate that S1 binding has a weak but detectable effect on the secondary structure of DsrA or rpoS mRNA.     

Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5'-leader

The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor sigmaS, includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of sigmaS, were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target.     

Regulation of RpoS by a novel small RNA: the characterization of RprA

Translational regulation of the stationary phase sigma factor RpoS is mediated by the formation of a double-stranded RNA stem-loop structure in the upstream region of the rpoS messenger RNA, occluding the translation initiation site. The interaction of the rpoS mRNA with a small RNA, DsrA, disrupts the double-strand pairing and allows high levels of translation initiation. We screened a multicopy library of Escherichia coli DNA fragments for novel activators of RpoS translation when DsrA is absent. Clones carrying rprA (RpoS regulator RNA) increased the translation of RpoS. The rprA gene encodes a 106 nucleotide regulatory RNA. As with DsrA, RprA is predicted to form three stem-loops and is highly conserved in Salmonella and Klebsiella species. Thus, at least two small RNAs, DsrA and RprA, participate in the positive regulation of RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. RprA is non-essential. Mutations in the gene interfere with the induction of RpoS after osmotic shock when DsrA is absent, demonstrating a physiological role for RprA. The existence of two very different small RNA regulators of RpoS translation suggests that such additional regulatory RNAs are likely to exist, both for regulation of RpoS and for regulation of other important cellular components.     

Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs

The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Qbeta replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5'-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq(-) strain. It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack.     

Interactions of the non-coding RNA DsrA and RpoS mRNA with the 30 S ribosomal subunit

Expression of sigma(s), the gene product of rpoS, is controlled translationally in response to many environmental stresses. DsrA, a small 87-nucleotide non-coding RNA molecule, acts to increase translational efficiency of RpoS mRNA under some growth conditions. In this work, we demonstrate that DsrA binds directly to the 30 S ribosomal subunit with an observed equilibrium affinity of 2.8 x 10(7) m(-1). DsrA does not compete with RpoS mRNA or tRNA(f)(Met) for binding to the 30 S subunit. The 5' end of DsrA binds to 30 S subunits with an observed equilibrium association constant of 2.0 x 10(6) m(-1), indicating that the full affinity of the interaction requires the entire DsrA sequence. In order to investigate translational efficiency of RpoS mRNA, we examined both ribosome-binding site accessibility and the binding of RpoS mRNA to 30 S ribosomal subunits. We find that that ribosome-binding site accessibility is modulated as a function of divalent cation concentration during mRNA renaturation and by the presence of an antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA fragment. The ribosome-binding site accessibility correlates with the amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.