The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA

Small noncoding RNAs (sRNAs) regulate the response of bacteria to environmental stress in conjunction with the Sm-like RNA binding protein Hfq. DsrA sRNA stimulates translation of the RpoS stress response factor in Escherichia coli by base-pairing with the 5′ leader of the rpoS mRNA and opening a stem–loop that represses translation initiation. We report that rpoS leader sequences upstream of this stem–loop greatly increase the sensitivity of rpoS mRNA to Hfq and DsrA. Native gel mobility shift assays show that Hfq increases the rate of DsrA binding to the full 576 nt rpoS leader as much as 50-fold. By contrast, base-pairing with a 138-nt RNA containing just the repressor stem–loop is accelerated only twofold. Deletion and mutagenesis experiments showed that sensitivity to Hfq requires an upstream AAYAA sequence. Leaders long enough to contain this sequence bind Hfq tightly and form stable ternary complexes with Hfq and DsrA. A model is proposed in which Hfq recruits DsrA to the rpoS mRNA by binding both RNAs, releasing the self-repressing structure in the mRNA. Once base-pairing between DsrA and rpoS mRNA is established, interactions between Hfq and the mRNA may stabilize the RNA complex by removing Hfq from the sRNA.     

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.     

Antagonistic functions between the RNA chaperone Hfq and an sRNA regulate sensitivity to the antibiotic colicin

The RNA chaperone Hfq is a key regulator of the function of small RNAs (sRNAs). Hfq has been shown to facilitate sRNAs binding to target mRNAs and to directly regulate translation through the action of sRNAs. Here, we present evidence that Hfq acts as the repressor of cirA mRNA translation in the absence of sRNA. Hfq binding to cirA prevents translation initiation, which correlates with cirA mRNA instability. In contrast, RyhB pairing to cirA mRNA promotes changes in RNA structure that displace Hfq, thereby allowing efficient translation as well as mRNA stabilization. Because CirA is a receptor for the antibiotic colicin Ia, in addition to acting as an Fur (Ferric Uptake Regulator)‐regulated siderophore transporter, translational activation of cirA mRNA by RyhB promotes colicin sensitivity under conditions of iron starvation. Altogether, these results indicate that Fur and RyhB modulate an unexpected feed‐forward loop mechanism related to iron physiology and colicin sensitivity.     

Unveiling the biotransformation mechanism of indole in a Cupriavidus sp. strain

Small RNAs (sRNAs), particularly those that act by limited base pairing with mRNAs, are part of most regulatory networks in bacteria. In many cases, the base‐pairing interaction is facilitated by the RNA chaperone Hfq. However, not all bacteria encode Hfq and some base‐pairing sRNAs do not require Hfq raising the possibility of other RNA chaperones. Candidates are proteins with homology to FinO, a factor that promotes base pairing between the FinP antisense sRNA and the traJ mRNA to control F plasmid transfer. Recent papers have shown that the Salmonella enterica FinO‐domain protein ProQ binds a large suite of sRNAs, including the RaiZ sRNA, which represses translation of the hupA mRNA, and the Legionella pneumophila protein RocC binds the RocR sRNA, which blocks expression of competence genes. Here we discuss what is known about FinO‐domain structures, including the recently solved Escherichia coli ProQ structure, as well as the RNA binding properties of this family of proteins and evidence they act as chaperones. We compare these properties with those of Hfq. We further summarize what is known about the physiological roles of FinO‐domain proteins and enumerate outstanding questions whose answers will establish whether they constitute a second major class of RNA chaperones.     

MicA sRNA links the PhoP regulon to cell envelope stress

Numerous small RNAs regulators of gene expression exist in bacteria. A large class of them binds to the RNA chaperone Hfq and act by base pairing interactions with their target mRNA, thereby affecting their translation and/or stability. They often have multiple direct targets, some of which may be regulators themselves, and production of a single sRNA can therefore affect the expression of dozens of genes. We show in this study that the synthesis of the Escherichia coli pleiotropic PhoPQ two‐component system is repressed by MicA, a σ^E‐dependent sRNA regulator of porin biogenesis. MicA directly pairs with phoPQ mRNA in the translation initiation region of phoP and presumably inhibits translation by competing with ribosome binding. Consequently, MicA downregulates several members of the PhoPQ regulon. By linking PhoPQ to σ^E, our findings suggest that major cellular processes such as Mg²⁺ transport, virulence, LPS modification or resistance to antimicrobial peptides are modulated in response to envelope stress. In addition, we found that Hfq strongly affects the expression of phoP independently of MicA, raising the possibility that even more sRNAs, which remain to be identified, could regulate PhoPQ synthesis.     

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.     

Modification of the RpoS network with a synthetic small RNA

Translation of the sigma factor RpoS is activated by DsrA, RprA and ArcA, three small non-coding sRNAs (sRNA) that expose the ribosome-binding site (RBS) by opening up an inhibitory loop. In the RpoS network, no sRNAs have been found to pair with the RBS, a most common sRNA target site in bacteria. Here, we generate Ribo-0, an artificial sRNA, which represses rpoS translation by pairing with the RBS. Ribo-0 bypasses the RNA chaperon Hfq but requires the RBS to be loosely blocked. Ribo-0 interacts with DsrA and reshapes the RpoS network. Specifically, in the intact RpoS network, DsrA activates rpoS translation by freeing up the RBS. In the modified RpoS network where Ribo-0 is introduced, the DsrA-caused RBS exposure facilitates Ribo-0 binding, thereby strengthening Ribo-0 inhibition. In other words, Ribo-0 changes DsrA from an activator to an accomplice for repressing rpoS translation. This work presents an artificial mechanism of rpoS regulation, reveals mutual effects of native and synthetic players and demonstrates genetic context-dependency of their functions.     

Hfq-bridged ternary complex is important for translation activation of rpoS by DsrA

The rpoS mRNA, which encodes the master regulator σ^S of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. It has recently been proposed that one mechanism underlying Hfq action is to bridge a transient ternary complex by simultaneously binding to rpoS and DsrA. However, no structural evidence of Hfq simultaneously bound to different RNAs has been reported. We detected simultaneous binding of Hfq to rpoS and DsrA fragments. Crystal structures of AU6A•Hfq•A7 and Hfq•A7 complexes were resolved using 1.8- and 1.9-Å resolution, respectively. Ternary complex has been further verified in solution by NMR. In vivo, activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex. This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA–mRNA annealing process.     

Translation inhibition from a distance: The small RNA SgrS silences a ribosomal protein S1-dependent enhancer

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA–mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5ʹ untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.