Hfq Binding Site Research Articles

Integrated Hfq-interacting RNAome and transcriptomic analysis reveals complex regulatory networks of nitrogen fixation in root-associated Pseudomonas stutzeri A1501.

Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Establishing a Multivariate Model for Predictable Antisense RNA-Mediated Repression.

Recent advances in our understanding of RNA folding and functions have facilitated the use of regulatory RNAs such as synthetic antisense RNAs (asRNAs) to modulate gene expression. However, despite the simple and universal complementarity rule, predictable asRNA-mediated repression is still challenging due to the intrinsic complexity of native asRNA-mediated gene regulation. To address this issue, we present a multivariate model, based on the change in free energy of complex formation (Δ GCF) and percent mismatch of the target binding region, which can predict synthetic asRNA-mediated repression efficiency in diverse contexts. First, 69 asRNAs that bind to multiple target mRNAs were designed and tested to create the predictive model. Second, we showed that the same model is effective predicting repression of target genes in both plasmids and chromosomes. Third, using our model, we designed asRNAs that simultaneously modulated expression of a toxin and its antitoxin to demonstrate tunable control of cell growth. Fourth, we tested and validated the same model in two different biotechnologically important organisms: Escherichia coli Nissle 1917 and Bacillus subtilis 168. Last, multiple parameters, including target locations, the presence of an Hfq binding site, GC contents, and gene expression levels, were revisited to define the conditions under which the multivariate model should be used for accurate prediction. Together, 434 different strain-asRNA combinations were tested, validating the predictive model in a variety of contexts, including multiple target genes and organisms. The result presented in this study is an important step toward achieving predictable tunability of asRNA-mediated repression.

The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs.

MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg2+ ions in Escherichia coli. MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, eptB and ygdQ, by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products. The data showed that the mutation on the rim of the Hfq ring trapped MgrR on Hfq preventing the annealing of MgrR to either mRNA. The mutation in the distal face slowed the ternary complex formation and affected the release of MgrR-mRNA complexes from Hfq, while the mutation in the proximal face weakened the MgrR binding to Hfq and in this way affected the annealing. Moreover, competition assays established that MgrR bound to both faces of Hfq and competed against other sRNAs. Further studies showed that uridine-rich sequences located in less structurally stable regions served as Hfq binding sites in each mRNA. Overall, the data show that the binding of MgrR sRNA to both faces of the Hfq ring enables it to efficiently anneal to target mRNAs. It also confers on MgrR a competitive advantage over other sRNAs, which could contribute to efficient cellular response to changes in magnesium homeostasis.

An RNA-dependent mechanism for transient expression of bacterial translocation filaments.

The prokaryotic RNA chaperone Hfq mediates sRNA–mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5′ UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.

High-Resolution, High-Throughput Analysis of Hfq-Binding Sites Using UV Crosslinking and Analysis of cDNA (CRAC).

Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA-protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein-RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin-antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.

Development of Design Rules for Reliable Antisense RNA Behavior in E. coli.

A key driver of synthetic biology is the development of designable genetic parts with predictable behaviors that can be quickly implemented in complex genetic systems. However, the intrinsic complexity of gene regulation can make the rational design of genetic parts challenging. This challenge is apparent in the design of antisense RNA (asRNA) regulators. Though asRNAs are well-known regulators, the literature governing their design is conflicting and leaves the synthetic biology community without clear asRNA design rules. The goal of this study is to perform a comprehensive experimental characterization and statistical analysis of 121 unique asRNA regulators in order to resolve the conflicts that currently exist in the literature. asRNAs usually consist of two regions, the Hfq binding site and the target binding region (TBR). First, the behaviors of several high-performing Hfq binding sites were compared, in terms of their ability to improve repression efficiencies and their orthogonality. Next, a large-scale analysis of TBR design parameters identified asRNA length, the thermodynamics of asRNA-mRNA complex formation, and the percent of target mismatch as key parameters for TBR design. These parameters were used to develop simple asRNA design rules. Finally, these design rules were applied to construct both a simple and a complex genetic circuit containing different asRNAs, and predictable behavior was observed in both circuits. The results presented in this study will drive synthetic biology forward by providing useful design guidelines for the construction of asRNA regulators with predictable behaviors.

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5′-untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5′-untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs.

Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching

Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5′-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

Positional Effects of AAN Motifs in rpoS Regulation by sRNAs and Hfq

The Escherichia coli stationary phase transcription factor RpoS is translated in response to small noncoding RNAs (sRNAs), which base pair with the rpoS mRNA leader. The bacterial Sm-like protein Hfq anneals sRNAs with their mRNA targets by simultaneously binding the mRNA and sRNA. Intriguingly, Hfq is recruited to the rpoS leader via AAN motifs far upstream of the sRNA. SHAPE (selective 2′-hydroxyl acylation and primer extension) chemical footprinting showed that the rpoS leader is divided into a far upstream domain, an Hfq binding domain, and a downstream inhibitory stem–loop containing the sRNA and ribosome binding sites. To investigate how Hfq promotes sRNA–mRNA base pairing from a distance, we deleted the natural AAN Hfq binding site, and we inserted artificial AAN binding sites at various positions in the rpoS leader. All the relocated AAN motifs restored tight Hfq binding in vitro, but only insertion at the natural position restored Hfq-dependent sRNA annealing in vitro and sRNA regulation of rpoS translation in vivo. Furthermore, U-rich motifs in the downstream inhibitory domain stabilized the rpoS mRNA–Hfq complex and contributed to regulation of rpoS expression. We propose that the natural Hfq binding domain is optimal for positive regulation because it recruits Hfq to the mRNA and allows it to act on incoming sRNAs without opening the inhibitory stem–loop when sRNA is absent.

Small RNA Modules Confer Different Stabilities and Interact Differently with Multiple Targets

Bacterial Hfq-associated small regulatory RNAs (sRNAs) parallel animal microRNAs in their ability to control multiple target mRNAs. The small non-coding MicA RNA represses the expression of several genes, including major outer membrane proteins such as ompA, tsx and ecnB. In this study, we have characterised the RNA determinants involved in the stability of MicA and analysed how they influence the expression of its targets. Site-directed mutagenesis was used to construct MicA mutated forms. The 5′linear domain, the structured region with two stem-loops, the A/U-rich sequence or the 3′ poly(U) tail were altered without affecting the overall secondary structure of MicA. The stability and the target regulation abilities of the wild-type and the different mutated forms of MicA were then compared. The 5′ domain impacted MicA stability through an RNase III-mediated pathway. The two stem-loops showed different roles and disruption of stem-loop 2 was the one that mostly affected MicA stability and abundance. Moreover, STEM2 was found to be more important for the in vivo repression of both ompA and ecnB mRNAs while STEM1 was critical for regulation of tsx mRNA levels. The A/U-rich linear sequence is not the only Hfq-binding site present in MicA and the 3′ poly(U) sequence was critical for sRNA stability. PNPase was shown to be an important exoribonuclease involved in sRNA degradation. In addition to the 5′ domain of MicA, the stem-loops and the 3′ poly(U) tail are also shown to affect target-binding. Disruption of the 3′U-rich sequence greatly affects all targets analysed. In conclusion, our results have shown that it is important to understand the “sRNA anatomy” in order to modulate its stability. Furthermore, we have demonstrated that MicA RNA can use different modules to regulate its targets. This knowledge can allow for the engineering of non-coding RNAs that interact differently with multiple targets.

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 functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3′ poly(U) tail

Hfq-dependent sRNAs contain, at least, an mRNA base-pairing region, an Hfq-binding site, and a Rho-independent terminator. Recently, we found that the terminator poly(U) of Escherichia coli sRNAs is essential for Hfq binding and therefore for riboregulation. In this study, we tried to identify additional components within Hfq-binding sRNAs required for efficient Hfq binding by using SgrS as a model. We demonstrate by mutational and biochemical studies that an internal hairpin and an immediately upstream U-rich sequence also are required for efficient Hfq binding. We propose that the functional Hfq-binding module of SgrS consists of an internal hairpin preceded by a U-rich sequence and a Rho-independent terminator with a long poly(U) tail. We also show that the Rho-independent terminator alone can act as a functional Hfq-binding module when it is preceded by an internal U-rich sequence. The 3' region of most known sRNAs share the features corresponding to either a double- or single-hairpin-type Hfq-binding module. We also demonstrate that increasing the spacing between the base-pairing region and the Hfq-binding module reduces or impairs the silencing ability. These findings allowed us to design synthetic Hfq-binding sRNAs to target desired mRNAs.

Multiple factors dictate target selection by Hfq-binding small RNAs

Hfq-binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base-pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non-targets among the mRNAs predicted to base pair with Hfq-binding sRNAs is poorly understood. Using the Hfq-binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base-pairing in single or multiple base-pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non-targets predicted to base pair with Spot 42 lacked an Hfq-binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq-binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non-target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs.

PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action

Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼108 M−1s−1) observed with Cy3-Hfq was followed by a slow transition (0.5 s−1) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.

Artificial trans -encoded small non-coding RNAs specifically silence the selected gene expression in bacteria

Recently, many small non-coding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become a powerful gene silencing tool in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described first the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural sRNAs in Gram-negative bacteria, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. The atsRNAs led to gene silencing and RNase E dependent degradation of target mRNA. We also designed a series of atsRNAs which targeted the toxic genes in Staphyloccocus aureus, but found no significant interfering effect. We established an effective method for specific gene silencing in Gram-negative bacteria.

An Upstream Hfq Binding Site in the fhlA mRNA Leader Region Facilitates the OxyS-fhlA Interaction

BackgroundTo survive, bacteria must be able to adapt to environmental stresses. Small regulatory RNAs have been implicated as intermediates in a variety of stress-response pathways allowing dynamic gene regulation. The RNA binding protein Hfq facilitates this process in many cases, helping sRNAs base pair with their target mRNAs and initiate gene regulation. Although Hfq has been identified as a critical component in many RNPs, the manner by which Hfq controls these interactions is not known.Methodology/Principal FindingsTo test the requirement of Hfq in these mRNA-sRNA complexes, the OxyS-fhlA system was used as a model. OxyS is induced in response to oxidative stress and down regulates the translation of fhlA, a gene encoding a transcriptional activator for formate metabolism. Biophysical characterization of this system previously used a minimal construct of the fhlA mRNA which inadvertently removed a critical element within the leader sequence of this mRNA that effected thermodynamics and kinetics for the interaction with Hfq.Conclusions/SignificanceHerein, we report thermodynamic, kinetic and structural mapping studies during binary and ternary complex formation between Hfq, OxyS and fhlA mRNA. Hfq binds fhlA mRNA using both the proximal and distal surfaces and stimulates association kinetics between the sRNA and mRNA but remains bound to fhlA forming a ternary complex. The upstream Hfq binding element within fhlA is similar to (ARN)x elements recently identified in other mRNAs regulated by Hfq. This work leads to a kinetic model for the dynamics of these complexes and the regulation of gene expression by bacterial sRNAs.

Positive regulation by small RNAs and the role of Hfq

Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5' leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an (AAN)(4) Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37 degrees C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA*mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA*mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.

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.

Hfq Is a Regulator of F-Plasmid TraJ and TraM Synthesis inEscherichia coli

The F plasmid of Escherichia coli allows horizontal DNA transfer between an F(+) donor cell and an F(-) recipient. Expression of the transfer genes is tightly controlled by a number of factors, including the following plasmid-encoded regulatory proteins: TraJ, the primary activator of the 33-kb tra operon, and the autoregulators TraM and TraY. Here, we demonstrate that the host RNA binding protein, Hfq, represses TraJ and TraM synthesis by destabilizing their respective mRNAs. Mating assays and immunoblot analyses for TraM and TraJ showed that transfer efficiency and protein levels increased in host cells containing a disruption in hfq compared to wild-type cells in stationary phase. The stability of transcripts containing a putative Hfq binding site located in the intergenic untranslated region between traM and traJ was increased in hfq mutant donor cells, suggesting that Hfq destabilizes these transcripts. Electrophoretic mobility shift assays demonstrated that Hfq specifically binds this region but not the antisense RNA, FinP, encoded on the opposite strand. Together, these findings indicate that Hfq regulates traM and traJ transcript stability by a mechanism separate from FinOP-mediated repression.