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.     

Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing

Hfq is a key regulator involved in multiple aspects of stress tolerance and virulence of bacteria. There has been an intriguing question as to how this RNA chaperone achieves two completely opposite functions--annealing and unwinding--for different RNA substrates. To address this question, we studied the Hfq-mediated interaction of fragments of a non-coding RNA, DsrA, with its mRNA target rpoS by using single-molecule fluorescence techniques. These experiments permitted us to observe the mechanistic steps of Hfq-mediated RNA annealing/unwinding at the single-molecule level, for the first time. Our real-time observations reveal that, even if the ring-shaped Hfq displays multiple binding sites for its interaction with RNA, the regulatory RNA and the mRNA compete for the same binding site. The competition makes the RNA-Hfq interaction dynamic and, surprisingly, increases the overall annealing efficiency by properly aligning the two RNAs. We furthermore reveal that when Hfq specifically binds to only one of the two RNAs, the unwinding process dominates over the annealing process, thus shedding a new light on the substrate selectivity for annealing or unwinding. Finally, our results demonstrate for the first time that a single Hfq hexamer is sufficient to facilitate sRNA-mRNA annealing.     

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure

DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli. DsrA activates the translation of RpoS while repressing the translation of H-NS. The RNA-binding protein Hfq is necessary for DsrA to function in vivo. Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known. One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs. To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro. Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation. We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA. In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding. Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA. In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.     

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.     

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.     

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.     

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.     

Translational repression by bacteriophage MS2 coat protein does not require cysteine residues.

Previous studies implicated cysteine residues in the translational repressor (i.e. RNA binding) activity of the coat protein of bacteriophage MS2. It has been proposed that a protein sulfhydryl forms a transient covalent bond with an essential pyrimidine in the translational operator by a Michael addition reaction. We have utilized codon-directed mutagenesis methods to determine the importance of each of the two coat protein cysteines for repressor function in vivo. The results indicate that cys46 can be replaced by a variety of amino acids without loss of repressor function. Cys101, on the other hand, is more sensitive to substitution. Most position 101 substitutions inactivate the repressor, but one (arginine) results in normal repressor activity. Although the possibility of a transient covalent contact between cys101 and RNA is not categorically ruled out, construction of double mutants demonstrates that cysteines are not absolutely required for translational repression by coat protein.     

Distinct and overlapping binding sites of Pseudomonas aeruginosa Hfq and RsmA proteins on the non-coding RNA RsmY

In Pseudomonas aeruginosa the Rsm system is involved in regulation of quorum-sensing and virulence gene expression. Our recent studies revealed that the stability and abundance of the non-coding RNA RsmY, which antagonizes the translational regulator RsmA, is dependent on Hfq. Here, we show that Hfq and RsmA bind concurrently to RsmY. Enzymatic probing of RsmY RNA in the presence of RsmA and Hfq verified the proposed -GGA- motifs as RsmA binding sites and located Hfq binding sites in single-stranded regions adjacent to stem-loop structures, respectively. We conclude that distinct binding of Hfq and RsmA on RsmY RNA permits RsmY-mediated RsmA titration upon binding to and stabilization of RsmY RNA by Hfq. In addition, we provide evidence that Hfq sequesters RNase E cleavage sites on RsmY, which explains the previously observed dependence of RsmY RNA stability on Hfq.     

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.     

Why base tautomerization does not cause errors in mRNA decoding on the ribosome

The structure of the genetic code implies strict Watson–Crick base pairing in the first two codon positions, while the third position is known to be degenerate, thus allowing wobble base pairing. Recent crystal structures of near-cognate tRNAs accommodated into the ribosomal A-site, however, show canonical geometry even with first and second position mismatches. This immediately raises the question of whether these structures correspond to tautomerization of the base pairs. Further, if unusual tautomers are indeed trapped why do they not cause errors in decoding? Here, we use molecular dynamics free energy calculations of ribosomal complexes with cognate and near-cognate tRNAs to analyze the structures and energetics of G-U mismatches in the first two codon positions. We find that the enol tautomer of G is almost isoenergetic with the corresponding ketone in the first position, while it is actually more stable in the second position. Tautomerization of U, on the other hand is highly penalized. The presence of the unusual enol form of G thus explains the crystallographic observations. However, the calculations also show that this tautomer does not cause high codon reading error frequencies, as the resulting tRNA binding free energies are significantly higher than for the cognate complex.     

The activation mechanism of Hsp26 does not require dissociation of the oligomer

Small heat shock proteins (sHsps) are molecular chaperones that specifically bind non-native proteins and prevent them from irreversible aggregation. A key trait of sHsps is their existence as dynamic oligomers. Hsp26 from Saccharomyces cerevisiae assembles into a 24mer, which becomes activated under heat shock conditions and forms large, stable substrate complexes. This activation coincides with the destabilization of the oligomer and the appearance of dimers. This and results from other groups led to the generally accepted notion that dissociation might be a requirement for the chaperone mechanism of sHsps. To understand the chaperone mechanism of sHsps it is crucial to analyze the relationship between chaperone activity and stability of the oligomer. We generated an Hsp26 variant, in which a serine residue of the N-terminal domain was replaced by cysteine. This allowed us to covalently crosslink neighboring subunits by disulfide bonds. We show that under reducing conditions the structure and function of this variant are indistinguishable from that of the wild-type protein. However, when the cysteine residues are oxidized, the dissociation into dimers at higher temperatures is no longer observed, yet the chaperone activity remains unaffected. Furthermore, we show that the exchange of subunits between Hsp26 oligomers is significantly slower than substrate aggregation and even inhibited in the presence of disulfide bonds. This demonstrates that the rearrangements necessary for shifting Hsp26 from a low to a high affinity state for binding non-native proteins occur without dissolving the oligomer.     

The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein.

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 sigma(s) subunit of RNA polymerase. Sigma(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 hfq-encoded 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.     

Global analysis of small RNA and mRNA targets of Hfq

Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.     

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.