Impact of Hfq on the Bacillus subtilis transcriptome.

The RNA chaperone Hfq acts as a central player in post-transcriptional gene regulation in several Gram-negative Bacteria, whereas comparatively little is known about its role in Gram-positive Bacteria. Here, we studied the function of Hfq in Bacillus subtilis, and show that it confers a survival advantage. A comparative transcriptome analysis revealed mRNAs with a differential abundance that are governed by the ResD-ResE system required for aerobic and anaerobic respiration. Expression of resD was found to be up-regulated in the hfq- strain. Furthermore, several genes of the GerE and ComK regulons were de-regulated in the hfq- background. Surprisingly, only six out of >100 known and predicted small RNAs (sRNAs) showed altered abundance in the absence of Hfq. Moreover, Hfq positively affected the transcript abundance of genes encoding type I toxin-antitoxin systems. Taken the moderate effect on sRNA levels and mRNAs together, it seems rather unlikely that Hfq plays a central role in RNA transactions in Bacillus subtilis.

Duplex formation between the sRNA DsrA and rpoS mRNA is not sufficient for efficient RpoS synthesis at low temperature.

At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor RpoS, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by Hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs). Both the DEAD box helicase CsdA and Hfq have been implicated in DsrA·rpoS duplex formation. Hfq binding to A-rich sequences in the rpoS leader has been suggested to restructure the mRNA, and thereby to accelerate DsrA·rpoS duplex formation, which, in turn, was deemed to free the rpoS rbs and to permit ribosome loading on the mRNA. Several experiments designed to elucidate the role of Hfq in DsrA-mediated translational activation of rpoS mRNA have been conducted in vitro. Here, we assessed RpoS synthesis in vivo to further study the role of Hfq in rpoS regulation. We show that RpoS synthesis was reduced when DsrA was ectopically overexpressed at 24 °C in the absence of Hfq despite of DsrA·rpoS duplex formation. This observation indicated that DsrA·rpoS annealing may not be sufficient for efficient ribosome loading on rpoS mRNA. In addition, a HfqG29A mutant protein was employed, which is deficient in binding to A-rich sequences present in the rpoS leader but proficient in DsrA binding. We show that DsrA·rpoS duplex formation occurs in the presence of the HfqG29A mutant protein at low temperature, whereas synthesis of RpoS was greatly diminished. RNase T1 footprinting studies of DsrA·rpoS duplexes in the absence and presence of Hfq or HfqG29A indicated that Hfq is required to resolve a stem-loop structure in the immediate coding region of rpoS mRNA. These in vivo studies corroborate the importance of the A-rich sequences in the rpoS leader and strongly suggest that Hfq, besides stabilizing DsrA and accelerating DsrA·rpoS duplex formation, is also required to convert the rpoS mRNA into a translationally competent form.

Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq.

In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)(15) and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq(65)) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.

Structural flexibility of RNA as molecular basis for Hfq chaperone function.

In enteric bacteria, many small regulatory RNAs (sRNAs) associate with the RNA chaperone host factor Q (Hfq) and often require the protein for regulation of target mRNAs. Previous studies suggested that the hexameric Escherichia coli Hfq (Hfq(Ec)) binds sRNAs on the proximal site, whereas the distal site has been implicated in Hfq-mRNA interactions. Employing a combination of small angle X-ray scattering, nuclear magnetic resonance and biochemical approaches, we report the structural analysis of a 1:1 complex of Hfq(Ec) with a 34-nt-long subsequence of a natural substrate sRNA, DsrA (DsrA(34)). This sRNA is involved in post-transcriptional regulation of the E. coli rpoS mRNA encoding the stationary phase sigma factor RpoS. The molecular envelopes of Hfq(Ec) in complex with DsrA(34) revealed an overall asymmetric shape of the complex in solution with the protein maintaining its doughnut-like structure, whereas the extended DsrA(34) is flexible and displays an ensemble of different spatial arrangements. These results are discussed in terms of a model, wherein the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq.

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq.

The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).