Presence Of Hfq Research Articles

Models of Hfq interactions with small non-coding RNA in Gram-negative and Gram-positive bacteria.

Hfq is required by many Gram-negative bacteria to chaperone the interaction between small non-coding RNA (sRNA) and mRNA to facilitate annealing. Conversely and despite the presence of Hfq in many Gram-positive bacteria, sRNAs in Gram-positive bacteria bind the mRNA target independent of Hfq. Details provided by the Hfq structures from both Gram-negative and Gram-positive bacteria have demonstrated that despite a conserved global structure of the protein, variations of residues on the binding surfaces of Hfq results in the recognition of different RNA sequences as well as the ability of Hfq to facilitate the annealing of the sRNA to the mRNA target. Additionally, a subset of Gram-negative bacteria has an extended C-terminal Domain (CTD) that has been shown to affect the stability of the Hfq hexamer and increase the rate of release of the annealed sRNA-mRNA product. Here we review the structures of Hfq and biochemical data that have defined the interactions of the Gram-negative and Gram-positive homologues to highlight the similarities and differences in the interactions with RNA. These interactions provided a deeper understanding of the how Hfq functions to facilitate the annealing of sRNA-mRNA, the selectivity of the interactions with RNA, and the role of the CTD of Hfq in the interactions with sRNA.

Differential Chromosome- and Plasmid-Borne Resistance of Escherichia coli hfq Mutants to High Concentrations of Various Antibiotics.

The Hfq protein is a bacterial RNA chaperone, involved in many molecular interactions, including control of actions of various small RNA regulatory molecules. We found that the presence of Hfq was required for survival of plasmid-containing Escherichia coli cells against high concentrations of chloramphenicol (plasmid p27cmr), tetracycline (pSC101, pBR322) and ampicillin (pBR322), as hfq+ strains were more resistant to these antibiotics than the hfq-null mutant. In striking contrast, production of Hfq resulted in low resistance to high concentrations of kanamycin when the antibiotic-resistance marker was chromosome-borne, with deletion of hfq resulting in increasing bacterial survival. These results were observed both in solid and liquid medium, suggesting that antibiotic resistance is an intrinsic feature of these strains rather than a consequence of adaptation. Despite its major role as RNA chaperone, which also affects mRNA stability, Hfq was not found to significantly affect kan and tet mRNAs turnover. Nevertheless, kan mRNA steady-state levels were higher in the hfq-null mutant compared to the hfq+ strain, suggesting that Hfq can act as a repressor of kan expression.This observation does correlate with the enhanced resistance to high levels of kanamycin observed in the hfq-null mutant. Furthermore, dependency on Hfq for resistance to high doses of tetracycline was found to depend on plasmid copy number, which was only observed when the resistance marker was expressed from a low copy plasmid (pSC101) but not from a medium copy plasmid (pBR322). This suggests that Hfq may influence survival against high doses of antibiotics through mechanisms that remain to be determined. Studies with pBR322Δrom may also suggest an interplay between Hfq and Rom in the regulation of ColE1-like plasmid replication. Results of experiments with a mutant devoid of the part of the hfq gene coding for the C-terminal region of Hfq suggested that this region, as well as the N-terminal region, may be involved in the regulation of expression of antibiotic resistance in E. coli independently.

Polyadenylation in E. coli: a 20 year odyssey.

Polyadenylation in E. coli: a 20 year odyssey.

Fluorescence reporters for Hfq oligomerization and RNA annealing.

Fluorescence spectroscopy is a sensitive technique for detecting protein-protein, protein-RNA, and RNA-RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy. We also present a detailed protocol for measuring the rate of annealing between a molecular beacon and a target RNA in the presence of Hfq using a stopped-flow spectrometer.

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.

Hfq proximity and orientation controls RNA annealing

Regulation of bacterial gene networks by small non-coding RNAs (sRNAs) requires base pairing with messenger RNA (mRNA) targets, which is facilitated by Hfq protein. Hfq is recruited to sRNAs and mRNAs through U-rich- and A-rich-binding sites, respectively, but their distance from the sRNA–mRNA complementary region varies widely among different genes. To determine whether distance and binding orientation affect Hfq’s chaperone function, we engineered ‘toy’ RNAs containing strong Hfq-binding sites at defined distances from the complementary target site. We show that RNA annealing is fastest when the distal face of Hfq binds an A-rich sequence immediately 3′ of the target. This recruitment advantage is lost when Hfq binds >20 nt away from the target, but is partially restored by secondary structure that shortens this distance. Although recruitment through Hfq’s distal face accelerates RNA annealing, tight binding of six Us to Hfq’s proximal face inhibits annealing. Finally, we show that ectopic A-rich motifs dramatically accelerate base pairing between DsrA sRNA and a minimal rpoS mRNA in the presence of Hfq, demonstrating that proximity and orientation predict the activity of Hfq on long RNAs.

The influence of Escherichia coli Hfq mutations on RNA binding and sRNA•mRNA duplex formation in rpoS riboregulation

The Escherichia coli RNA binding protein Hfq plays an important role in regulating mRNA translation through its interactions with small non-coding RNAs (sRNAs) and specific mRNAs sites. The rpoS mRNA, which codes for a transcription factor, is regulated by several sRNAs. DsrA and RprA enhance translation by pairing to a site on this mRNA, while OxyS represses rpoS mRNA translation. To better understand how Hfq interacts with these sRNAs and rpoS mRNA, the binding of wt Hfq and eleven mutant Hfqs to DsrA, RprA, OxyS and rpoS mRNA was examined. Nine of the mutant Hfq had single-residue mutations located on the proximal, distal, and outer-edge surfaces of the Hfq hexamer, while two Hfq had truncated C-terminal ends. Hfq with outer-edge mutations and truncated C-terminal ends behaved similar to wt Hfq with regard to binding the sRNAs, rpoS mRNA segments, and stimulating DsrA• rpoS mRNA formation. Proximal surface mutations decreased Hfq binding to the three sRNAs and the rpoS mRNA segment containing the translation initiation region. Distal surface mutations lowered Hfq's affinity for the rpoS mRNA segment containing the (ARN) 4 sequence. Strong Hfq binding to both rpoS mRNA segments appears to be needed for maximum enhancement of DsrA• rpoS mRNA annealing. OxyS bound tightly to Hfq but exhibited weak affinity for rpoS mRNA containing the leader region and 75 nt of coding sequence in the absence or presence of Hfq. This together with other results suggest OxyS represses rpoS mRNA translation by sequestering Hfq rather than binding to rpoS mRNA.

Compartmentalization in a Water-in-Oil Emulsion Repressed the Spontaneous Amplification of RNA by Qβ Replicase

During RNA replication mediated by Qbeta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target RNA replication difficult, especially for long RNA such as genomic RNA of Qbeta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication in the presence of host factor Hfq has not been reported in vitro. Here, we report a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qbeta replicase. This method allowed us to amplify the phage Qbeta genome RNA exponentially without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the study of genomic RNA replication without special care for the undesired RQ RNA amplification.

Identification of small Hfq-binding RNAs in Listeria monocytogenes

The RNA-binding protein Hfq plays important roles in bacterial physiology and is required for the activity of many small regulatory RNAs in prokaryotes. We have previously shown that Hfq contributes to stress tolerance and virulence in the Gram-positive human pathogen Listeria monocytogenes. In the present study, we performed coimmunoprecipitations followed by enzymatic RNA sequencing to identify Hfq-binding RNA molecules in L. monocytogenes. The approach resulted in the discovery of three small RNAs (sRNAs). The sRNAs are conserved between Listeria species, but were not identified in other bacterial species. The initial characterization revealed a number of unique features displayed by each individual sRNA. The first sRNA is encoded from within an annotated gene in the L. monocytogenes EGD-e genome. Analogous to most regulatory sRNAs in Escherichia coli, the stability of this sRNA is highly dependent on the presence of Hfq. The second sRNA appears to be produced by a transcription attenuation mechanism, and the third sRNA is present in five copies at two different locations within the L. monocytogenes EGD-e genome. The cellular levels of the sRNAs are growth phase dependent and vary in response to growth medium. All three sRNAs are expressed when L. monocytogenes multiplies within mammalian cells. This study represents the first attempt to identify sRNAs in L. monocytogenes.

The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation.

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.

Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding

The adaptation of mRNA stability to environmental changes is a means of cells to adjust the level of gene expression. The Escherichia coli ompA mRNA has served as one of the paradigms for regulated mRNA decay in prokaryotes. The stability of the transcript is known to be correlated inversely with the bacterial growth rate. Thus, the regulation of ompA mRNA stability meets the physiological needs to adjust the level of ompA expression to the rate of cell division. Recently, host factor I (Hfq/HF1) was shown to be involved in the regulation of ompA mRNA stability under slow growth conditions. Here, we present the first direct demonstration that 30S ribosomes bound to the ompA 5'-UTR protect the transcript from RNase E cleavage in vitro. However, the 30S protection was found to be abrogated in the presence of Hfq. Toeprinting and in vitro translation assays revealed that translation of ompA is repressed in the presence of Hfq. These in vitro studies are corroborated by in vivo expression studies demonstrating that the reduced synthesis rate of OmpA effected by Hfq results in functional inactivation of the ompA mRNA. The data are discussed in terms of a model wherein Hfq regulates the stability of ompA mRNA by competing with 30S ribosomes for binding to the ompA 5'-UTR.

Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I.

Current evidence suggests that the length of poly(A) tails of bacterial mRNAs result from a competition between poly(A) polymerase and exoribonucleases that attack the 3' ends of RNAs. Here, we show that host factor Hfq is also involved in poly(A) tail metabolism. Inactivation of the hfq gene reduces the length of poly(A) tails synthesized at the 3' end of the rpsO mRNA by poly(A) polymerase I in vivo. In vitro, Hfq stimulates synthesis of long tails by poly(A) polymerase I. The strong binding of Hfq to oligoadenylated RNA probably explains why it stimulates elongation of primers that already harbor tails of 20-35 A. Polyadenylation becomes processive in the presence of Hfq. The similar properties of Hfq and the PABPII poly(A) binding protein, which stimulates poly(A) tail elongation in mammals, indicates that similar mechanisms control poly(A) tail synthesis in prokaryotes and eukaryotes.