- Email: [email protected]
Hfq structure, function and ligand binding
Hfq structure, function and ligand binding Richard G Brennan and Todd M Link Recent studies on Hfq have provided a deeper understanding of the multiple functions of this pleiotropic post-transcriptional regulator. Insights into the mechanism of Hfq action have come from a variety of approaches. A key finding was the characterization of two RNA binding sites: the Proximal Site, which binds sRNA and mRNA; and the Distal Site, which binds poly(A) tails. Hfq was shown to interact with PAP I, PNP and RNase E, proteins that are involved in mRNA decay and in vitro, was shown to form fibres, the physiological significance of which is unknown. Fluorescence resonance energy transfer (FRET) studies directly demonstrated the role of Hfq as a chaperone that facilitates the interaction between sRNAs and target mRNAs. There are still, however, some unresolved questions. Addresses Department of Biochemistry and Molecular Biology, Unit 1000 University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard Houston, TX 77030-4009, USA Corresponding author: Brennan, Richard G ([email protected])
Current Opinion in Microbiology 2007, 10:125–133 This review comes from a themed issue on Cell regulation Edited by Gisela Storz and Dieter Haas Available online 28th March 2007 1369-5274/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.03.015
Introduction Escherichia coli Hfq (host factor required for phage Qb RNA replication, also known as host factor 1) is a 102 amino acid residue (11.2 kDa) protein that was first identified as an endogenous bacterial protein which, along with the ribosomal protein S1, was necessary for the replication of the RNA plus-strand of bacteriophage Qb [1–3]. Subsequent phylogenetic analyses revealed the presence of Hfq orthologues in approximately half of all sequenced Gram-positive and Gram-negative bacteria, with several bacteria such as Bacillus anthracis encoding more than one hfq gene [4,5]. At least one archaeon, Methanococcus jannaschii, contains a protein that is related to Hfq. The Hfq family of proteins are thermostable, range in length from 70 to 110 amino acid residues and form homohexamers [2,5–7]. In E. coli, Hfq is highly abundant with an estimated 50 000 to 60 000 copies (10 000 www.sciencedirect.com
hexamers) per cell, of which 80% to 90% are found in the cytoplasmic fraction in association with ribosomes [8]. Given the role of in the replication of the Qb RNA virus and its association with ribosomes, early studies showed that this protein bound RNA and displayed its highest affinities for poly(A) and single-stranded AU-rich tracts [9–11]. More recently, the latter preference has been refined, whereby Hfq has been shown to bind with greater avidity to RNA sites containing a short, single-stranded stretch of uridines and adenosines, which are either preceded or followed by a stem-loop structure [12– 14,15]. The remaining intracellular Hfq is associated with the nucleoid and was reported to bind intrinsically curved DNA using phased A-tracts [16]. By contrast, Hfq does not bind the shorter single-stranded oligodeoxyribonucleotide dA7 [17]. Beyond Qb replication, the innate importance of Hfq in cellular function became clear when an hfq null mutant was constructed in E. coli [18]. This mutant showed pleiotropic effects, including decreased growth rates, increased sensitivity to ultraviolet light, mutagens and oxidants and increased cell length. Moreover, an hfq mutation activates or suppresses the synthesis of more than 50 proteins, due in part to the requirement of Hfq for the efficient translation of the rpoS gene, which encodes ss, the sigma factor expressed under a variety of stress conditions and in stationary phase [19–21]. One of the first connections between the ability of Hfq to bind small, noncoding RNAs (sRNAs) and its role in post-transcriptional regulation became evident in studies on OxyS, a 109 nucleotide regulator of the oxidative stress response. This sRNA was shown to bind directly to Hfq and thereby increase OxyS RNA interaction with rpoS mRNA as part of its mechanism to inhibit rpoS translation [5,22]. The now-observed wide-spread use of Hfq in RNA metabolic processes is underscored by the finding that 22 sRNAs of E. coli bind Hfq, a probable requirement in order for them to carry out their cellular functions through antisense recognition of target mRNAs [23]. Clearly the involvement of Hfq in post-transcriptional regulation will surely grow as methods to detect novel sRNAs improve [24].
Hfq as a post-transcriptional regulator Hfq carries out its post transcriptional regulatory function of several genes by binding trans-encoded sRNAs and perhaps their mRNA targets in order to facilitate their direct interaction. Hence, Hfq is a riboregulator and, ever since these early studies, it has been shown to interact functionally with over a dozen sRNAs (the subject of many of the reviews in this issue) [22,23,25,26]. This Current Opinion in Microbiology 2007, 10:125–133
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ultimately enables stabile sRNA–mRNA hybrid duplexes to form through small stretches of complementary sequences. One outcome of such binding is the removal of a translation-inhibitory structure that subsequently enables ribosome access to the transcription initiation region or the creation of an sRNA–mRNA structure that blocks ribosome access to that region. In its riboregulatory role, Hfq has been called an RNA chaperone because nuclease digestion and protection experiments and hydroxy radical footprinting of some sRNAs (e.g. OxyS and Spot 42) and mRNAs (e.g. sodB and ompA) have shown significant protection pattern differences arising from the binding of Hfq indicating that Hfq-binding changes their structures [13,27]. In further support of this designation as a chaperone, Hfq can dissociate or be proteolytically removed from target mRNA or sRNA– mRNA complex, yet the newly formed structures are stabile [13,28,29]. By contrast Hfq does not alter the secondary structure of DsrA, at least not significantly, but more probably changes the tertiary structure and brings about sRNA–mRNA base pairing by increasing the local concentration of the regulatory RNA and its target [12]. Regardless, both mechanisms (active reconfiguration of structure or simple concentration of sRNA– mRNA targets) would bring about the formation of the sRNA–mRNA complex. In addition to regulating translation in an sRNAdependent manner, Hfq modulates the decay of some mRNAs, (e.g. rpsO, which encodes for ribosomal protein S15) by binding to their poly(A) tails, stimulating poly(A) adenylation by poly(A) polymerase I (PAP I) and protecting this message from polynucleotide phosphorylase (PNP), RNase II and RNase E, enzymes involved in mRNA degradation [30–33]. Hfq has also been shown to modulate by two distinct mechanisms the half-life of ompA mRNA, which encodes the major outer membrane protein of E. coli [34,35]. Perhaps not unexpectedly, Hfq also has been found to autoregulate its own expression at the translational level by binding two sites on the hfq message thereby inhibiting the formation of the translational initiation complex [36]. The goal of this opinion piece is to briefly review the known structures of Hfq, to focus on the latest Hfq–RNA binding studies and, in combination with some older studies, to propose a structural, albeit speculative, model for Hfq–poly(A) binding, and to describe results revealing interactions between Hfq and components of the degradosome, as well as Hfq fibre formation.
Hfq is an Sm Protein Perhaps one of the largest remaining hurdles in fully understanding Hfq function is the lack of relevant high-resolution structures. Regardless, the four described Hfq structures — three apoproteins and one with bound RNA — have provided invaluable insight into the RNA Current Opinion in Microbiology 2007, 10:125–133
binding mechanisms of Hfq, either directly or in conjunction with site-directed mutagenesis studies [14,15,37,38]. The apoprotein structures include Hfq from Staphylococcus aureus [17], E. coli (residues 1–72) [39] and most recently Pseudomonas aeruginosa [40]. The structure of the only Hfq–RNA complex is that of S. aureus Hfq bound to the hepta-oligoribonucleotide, AU5G [17]. All structures revealed unequivocally that Hfq is an Sm protein. Sm and Sm-like proteins are found in eukaryotes and archaea and are involved in multiple aspects of RNA metabolism including splicing and mRNA decay [41]. These proteins are characterized by two highly conserved regions, the so-named Sm1 and Sm2 motifs [42]. The early difficulty in assigning Hfq to the Sm family was the lack of sequence homology in the Sm2 motif. However, by 2002 several groups had correctly included Hfq proteins in the greater Sm and Smlike superfamily [4,6,7,43]. Hfq forms a toroid with an outer diameter of 70 A˚ and a ‘thickness’ of 25 A˚. The central pore is 8 A˚ to 12 A˚ wide in the apoprotein structure. The structures of all Hfq proteins are characterized by an N-terminal a helix followed by five b strands that form a highly bent sheet that display the topology b5a1b1b2b3b4 (Figure 1). The Sm1 motif encompasses the first three b strands, whereas the Sm2 motif is composed of b strands 4 and 5. The cyclic hexamer is formed primarily by interactions between residues from b4 and b5 from the apposing subunit. The lone a helix sits on top of the sheet on what is now referred to as the ‘distal side’. The subunit structures of Hfq and Sm proteins are very similar and display root mean squared deviations ranging from 0.37 A˚2 to 0.52 A˚2 (Hfq to Hfq comparison) and 0.85 A˚2 to 1.3 A˚2 (Hfq to archaeal Lsm and Human Sm comparison) [39]. Thus, whether these proteins are hexamers or heptamers impinges little on the structures of their individual subunits. Intriguingly Hfq has been reported to have an ATPase activity, albeit relatively weak [44]. Sequence comparison between Hfq and the heat shock protein, ClpB, revealed some homology between the ATP binding site of ClpB and a stretch of Hfq sequence that includes b2 (Figure 1). This ‘modified Walker A box’ is rather open, and how and where ATP binds, and what is the role of the ATPase activity are under investigation.
Hfq binds sRNA and AU-rich sequences on the proximal side The structure of the S. aureus Hfq in complex with the hepta-oligoribonucleotide, AU5G, has provided initial information on how uridine-rich stretches bind, as well as information on one mechanism of adenosine binding (Figure 2) [17]. The major contributors to RNA binding are located in the Sm1 and Sm2 motifs with residues on the loops between b2 and b3 and b4 and b5 playing a central role. The RNA expands and fills the central, basic pore on the ‘proximal side’ of the hexamer and binds in a www.sciencedirect.com
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Figure 1
Overlay of the structures of four bacterial Hfq proteins. The Hfq structures of P. aeruginosa (PDB code 1U1T), E. coli (PDB code 1HK9), and S. aureus with (PDB code 1KQ2) and without RNA (PDB code 1KQ1) are depicted as ribbons and colored cyan, salmon, yellow and magenta, respectively. The view is into the ‘Proximal’ face. The bound RNA, AU5G, is shown as solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored white, blue, red and orange, respectively. The dashed line envelopes one monomer of the hexameric protein with the b sheets colored magenta and the single a helix in cyan. The secondary structures are labeled for this monomer, as are the b strands b40 and b50 , which form the majority of the inter-subunit interface. The ‘modified Walker A box’, which is proposed to be involved in ATP binding and would encompass strand b2 [44], is labeled on one subunit and shown in blue.
circular manner except for the 30 guanosine, which is located at the entrance of the pore (Figures 1 and 3a). Each of the six potential AU nucleotide binding pockets is composed of residues from two adjacent subunits. Both uridine and adenosine bind similarly, whereby their nucleobases stack against the aromatic side chain of residue Tyr42 and the same residue from a neighboring subunit (Figure 2). This residue is a phenylalanine in the Hfqs of E. coli and P. aeruginosa and identical base-side chain stacking interactions are expected. Other residues involved in binding include Sm1 residue Lys41, which uses its main-chain carbonyl oxygen atom to interact with N1 and N9 atoms of the uracil and adenine rings, respectively, as well as its side-chain nitrogen to hydrogen bond to the O4 oxygens of some uracils. The methylene side chain of residue Lys41 also stacks with some uracils. Sm2 residue Lys57, which is part of the highly conserved KH motif found in all Hfq proteins, makes a hydrogen bond to the uracil O2 atom, whilst Sm2 residue His58 interacts www.sciencedirect.com
Figure 2
Hfq–RNA interactions in the Proximal Site. These contacts are observed in the S. aureus Hfq–AU5G crystal structure (1KQ1). The cyan and yellow coloring of residues and ribbon indicates one of two neighboring subunits that form the binding pocket for each nucleotide. Residues are numbered using the S. aureus Hfq protein sequence. (a) Uridine interactions. The repeating pUp unit is emphasized as solid sticks. (b) Adenosine interactions. The terminal Ap is emphasized by solid sticks. The side chain nitrogen and oxygen atoms are colored blue and red, respectively. The nucleotide nitrogen, oxygen, carbon and phosphorus atoms are colored blue, red, grey and orange, respectively.
with the phosphate group and the O20 of some ribose rings. The latter interaction could be used to favor RNA binding over DNA binding. Unexpectedly, residue Gln8, which is outside the Sm motifs and located on helix a1, is also involved. The side chain of this residue ‘reads’ the uracil and adenine bases and with residues Lys41 and Lys57 is involved in discrimination against cytidine and guanosine binding. The S. aureus Hfq-AU5G structure is informative for certain key aspects of RNA binding, including how short tracts of AU-rich single-stranded RNA can interact with this riboregulator. Of course, one structure does not tell the entire story, and several points must be made. First, given that most Hfq binding sites on their target sRNAs contain stretches shorter than six single-stranded uridines or adenosines that are often interrupted by cytidines and guanosines, it is unlikely that all six binding pockets on the proximal side (or face) are filled simultaneously by a single RNA. Second, these pockets are clearly not the only RNA binding sites on this face as work by the groups of Sledjeski, Feig and Wartell [12,14,15] has demonstrated that other residues of the proximal face, at least in E. coli, as well as other elements of sRNAs, that is their stems and loops, are involved in binding. Interestingly, one of these studies also indicated that residues on the distal face contributed to rpoS mRNA binding [14]. Furthermore, hfq point mutations at positions 3 and 17, which precede the Sm1 motif, cannot support Qb replication, whereas those at Current Opinion in Microbiology 2007, 10:125–133
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Figure 3
Electrostatic potential energy surfaces of the known and proposed RNA binding sites of the S. aureus and E. coli Hfq proteins. (a–c) Views of the proximal side and distal electrostatic surfaces of S. aureus Hfq, respectively. (d–f) Views of the proximal, side and distal electrostatic surfaces of E. coli Hfq, respectively (blue is electropositive and red is electronegative). The side view of E. coli Hfq includes a plausible RNA binding cleft that would enable A27 to bind to both the Proximal and Distal Sites. The view of the Distal Site of the E. coli Hfq shows a possible binding site for A18 (i.e. a poly(A) tail). The bound RNAs are shown as solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored white, blue, red and orange, respectively. The electrostatic potential energy surfaces were created by PyMol and the APBS plug-in (Delano Scientific LLC, Palo Alto, CA).
residue 56, (Lys57 in S. aureus; Figure 2) can, implying Qb-binding does not use at least some highly conserved residues of the proximal pore to bind [37]. Intriguingly, a second, ‘external’ RNA binding site, which used residues of the N-terminal a helix and aromatic residues of b2, was observed in the crystal structure of the archaeaon Pyrococcus abyssi Sm core, bound to RNA [45]. Third, the Hfq–AU5G structure suggests that single-stranded RNA might be able to traverse the central pore of the proximal face to reach the Current Opinion in Microbiology 2007, 10:125–133
distal face (Figure 1). In support, the pore has a positive electrostatic surface and, upon RNA binding, has expanded to 15 A˚ wide, which is wide enough to thread a single strand through to the other side (Figures 1 and 3a,d). A 10 A˚ resolution structure of the U1-snRNP indicates RNA does pass through the hole of the Sm heptamer [46]. However, the assembly processes of the heptameric Sm proteins and the hexameric Hfq proteins are quite different. Subsets of Sm subunits assemble on the www.sciencedirect.com
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RNA whereas Hfq proteins are always hexameric. Furthermore, the electrostatic surface of the distal side is fairly negative, which would disfavor threading (Figure 3c,d,f). Of course, RNA threading could require one or more divalent cations, which would neutralize this feature of the pore on the distal face. Clearly more structural, biochemical and in vivo studies will be necessary to clarify these points.
Figure 4
Hfq binds poly(A) tails on the distal side The early studies of de Haseth and Uhlenbeck [10] revealed that the affinity of Hfq for poly(A) RNA displayed a strong length and geometry dependence, in which linear sequences between 15 (pA15) and 27 (pA27) showed extremely tight binding and those of cyclized (pAN) showed higher relative affinity to their linear counterparts when N 18, but lower relative affinity when N 15 [10]. This suggested that the poly(A) binding site on the Hfq hexamer was circular. However, these data could not be reconciled with the AU5G binding site. Recent studies to address the question of the poly(A) binding site on Hfq have provided intriguing results and a likely solution [14,15]. Using poly(A) stretches of either 18 (A18) or 27 (A27), these later studies revealed that residues Tyr25, Ile30 and Lys31 were involved in RNA binding, as their substitution by alanine reduced the affinity of E. coli Hfq for these poly(A)s fivefold to greater than 100-fold. More to the point, these residues are located on the distal side of Hfq, that is, on the side opposite the AU5G binding site. Alanine substitutions of residues Phe42, Tyr55, K56 and H57 on the proximal side, showed only twofold to threefold effects. Furthermore, A27 did not compete with DsrA binding to wild type Hfq, but rather resulted in a supershift. When these data are combined with some RNA stereochemical constraints and the cationic nature of the E. coli Hfq core (residues 1 to 68, pI 10), a plausible, albeit hypothetical, model of poly(A) binding to Hfq can be constructed (Figures 3e,f and 4). First, by contrast to the distal face of the S. aureus Hfq, which does not bind poly(A) with high affinity (TM Link and RG Brennan, unpublished), that of E. coli Hfq is exceedingly positive (Figure 3c,f). Second, examination of the distal face of the E. coli Hfq hexamer reveals that the side chains of residues Tyr25, Ile30 and Leu32 form a hydrophobic pocket (Figure 4). Moreover, the side chain of Lys31 on the adjacent b strand is pointing its positively charged amino group into the solvent, but stacking with the aromatic side chain of Tyr25. Third, the distance between the hydroxyl groups of residue Tyr25 of each subunit is 17 A˚. The distance that a trinucleotide repeat (A3) would span in the C30 endo conformation, which is that favored by RNA, is 17.7 A˚ (3 5.9 A˚). Thus, only a very slight adjustment in the RNA backbone or the Tyr25 side chain would enable one adenine nucleotide of a www.sciencedirect.com
Model of a hypothetical interactions between one adenine of a poly(A) RNA and Hfq residues of the E. coli Distal Site. These residues have been shown to affect poly(A), but not sRNA, polyU or AU5G binding to Hfq. Residues, shown as solid sticks, and secondary structures, shown as ribbons, from apposing subunits that form the putative RNA binding pocket are colored cyan and yellow. Nitrogen and oxygen atoms of each residue are colored blue and red, respectively. The bound poly(A) RNA is shown in solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored grey, blue, red and orange, respectively.
triplet to use each pocket. Moreover, residues Gln52 and Asn28, which are highly conserved amongst Hfq proteins, are nearby to read the hydrogen bond donors and acceptors of the adenine ring, and residue Lys31 is in position to interact with the phosphate backbone (Figure 4). Coincidentally A18 in the C30 endo conformation would occupy the entire site nearly perfectly (18 nucleotides 5.9 A˚ = 106.2 A˚; the circular distance starting and ending at the hydroxyl oxygen of any Tyr25 subunit is 6 17 A˚ = 102 A˚). Such a binding site might also explain the need for longer poly(A) tracts for highaffinity binding as the Hfq-adenosine interactions provided by individual pockets are probably insufficient until a more cooperative binding mode can be generated by longer A-tracts. Again, this model is quite speculative and ultimately, a structure of Hfq bound to poly(A) will be necessary to provide the details of this protein–RNA interaction. Given the stereochemistry, length and very basic nature of the distal face, one could speculate that this side of Hfq might well bind DNA A-tracts [16], which are found Current Opinion in Microbiology 2007, 10:125–133
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periodically throughout the E. coli chromosome, and thus in the nucleoid, and are associated with intrinsic DNA curvature [47]. Another notable feature of E. coli Hfq is the positive electrostatic surface of the trough that connects the proximal and distal faces, which again is in sharp contrast to the same area on the S. aureus Hfq, which shows a negative electrostatic surface (Figure 3b,e). Residues Arg16 and Arg17 of helix a1, Arg19 and Lys47 are the major contributors to the cationic nature of the trough. Interestingly, residue Arg16 has been implicated in DsrA binding [12,15] and residues Arg17 and Arg19 are located near the external U7 binding site of Lsm1 [45]. How other sRNAs, as well as DsrA and target RNAs use this site on Hfq will require additional studies.
between the sRNA and target mRNA, which results in the exposure of the rpoS ribosome binding site. Strandexchange takes place as a consequence of the rapid Hfq-mediated association of DsrA and rpoS, followed by slow melting of the rpoS stem region and similarly slow annealing of the two RNAs. Interestingly, these studies also revealed that Hfq disrupts preformed DsrA–rpoS complexes, albeit slowly. The physiological role of such disruption by Hfq is unclear, but could be part of the fine-tuning mechanism of sRNA regulation. It should be noted that these chaperoning activities of Hfq are independent of ATP hydrolysis thereby leaving the function of the ATPase activity of Hfq an open question. Similar FRET studies on other Hfq-regulated systems and those that attempt to measure donor–acceptor distances, should yield new insights into Hfq function.
Hfq–RNA stoichiometry
Hfq interacts with other proteins
Three recent studies have reported rather different results regarding just how many Hfq hexamers bind to one DsrA sRNA, one rpoS mRNA or one A18 molecule. In one study, isothermal titration calorimetric experiments revealed 1:1 (DsrA:Hfq hexamer) and (rpoS:Hfq hexamer) binding stoichiometries but a 2:1 (A18:Hfq hexamer) where A18 binds two identical sites [14]. The 1:1 rpoS: Hfq hexamer stoichiometry found by Mikulecky et al. [14] differs from the 1:2 (rpoS:Hfq hexamer) stoichiometry reported by Lease and Woodson [28], who determined this parameter by gel shift assays. Gel-shift experiments performed by Sun and Wartell [15] showed a 1:2 (DsrA:Hfq hexamer) stoichiometry, similar to that reported by Lease and Woodson. By contrast, fluorescence anisotropy studies on the Hfq binding to A18 show the 2:1 (A18:Hfq hexamer) stoichiometry, which appears to reflect two separate, but nearly equal, Hfq binding events on this poly(A) [15]. The reported differences are somewhat baffling, but could be the result of several factors, such as the different protein purification protocols, and hence their different specific activities, experimental methodologies and conditions, and the purity and homogeneity of their RNA samples. Structural data would be helpful in reconciling these data. In addition to crystallographic studies, single-particle image reconstruction could be a good avenue of pursuit. It would also be interesting to see how Hfq binding to any of its other protein partners influences stoichiometry. Ultimately, we might find that the in vivo stoichiometry is not fixed but rather flexible depending upon the cellular environment.
Given the riboregulatory functions of Hfq, it would be surprising if Hfq did not interact with components of the ribosome, degradosome or other cellular machines that are involved in RNA metabolism. Indeed, S1 has been shown to mediate Hfq binding to RNA polymerase and thereby modulate the transcriptional activity of the enzyme [44]. However, the demonstration of a direct protein–protein interaction between S1 and Hfq awaits. In a more recent study on the role of Hfq in polyadenylation-dependent mRNA decay, Kushner and colleagues [31] found that Hfq copurified with PNP and PAP I, two components of the degradosome, in an sRNA-independent manner. Further, that investigation revealed that Hfq could form a complex with PNP separately. Whether Hfq interacts directly with PAP I alone was not shown. Regardless, an Hfq–PNP–PAP I complex has clear physiological implications for the role of Hfq in regulating the polyadenylation of full length mRNAs that contain Rhoindependent transcription terminators. Hfq could destabilize the terminator stems, which are then direct targets of PAP I activity and ultimately subject to degradation by PNP and RNase II.
Hfq is a chaperone In studies that examined the chaperone activity of Hfq, Ha and colleagues [48] used real-time fluorescence resonance energy transfer (FRET) to monitor the effect of Hfq on promoting intermolecular base pairing between DsrA and rpoS. These studies revealed that Hfq accelerates strand exchange and subsequent annealing Current Opinion in Microbiology 2007, 10:125–133
In studies on the role of RNase E in the degradation of target mRNAs of the sRNAs, SgrS and RhyB, Aiba and coworkers [49,50] found that Hfq together with SgrS and RhyB copurifed with RNase E to form specialized ribonucleoprotein complexes that were distinct from the degradosome, that is PNP and RhlB RNA helicase, were not present. Enolase, another component of the degradosome that plays a crucial role in the rapid decay of ptsG mRNA, the target of SgrS, was also not present [51]. This work identified the C-terminal scaffold domain of RNase E as the Hfq-binding site. This domain had been found previously to participate in RhyB-mediated degradation of target mRNAs [52]. Binding of sRNA to Hfq does not appear necessary for the formation of a stabile Hfq– RNase E complex. Moreover, there is competition between Hfq and the degradosome components, except www.sciencedirect.com
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enolase, for binding to the C-terminal scaffold of RNase E, implying overlapping binding sites. Ultimately, the RNase E–Hfq–sRNA complex results in translational repression and rapid target mRNA degradation. Interestingly, in the absence of RNase E, Hfq binding to these sRNAs still results in translational repression, which appears to be sufficient for the gradual destabilization and destruction of the target mRNAs [50]. One other protein has been shown unequivocally, at least in vitro, to interact with Hfq. This is Hfq itself [53]. Using biophysical approaches and electron microscopy and image analysis at a resolution of 23 A˚, Hfq was shown to form well-ordered fibres that resemble those seen previously in archaeal Sm proteins (SmAPs) [54]. The helical pitch of the Hfq fibrillar structure is 245 A˚ and the corresponding vertical spacing between consecutive helical subunits is 40 A˚. These parameters result in 36 Hfq subunits per helical pitch. The fibres differ in detail with those of SmAPs, forming polar tubes and those of Hfq assembling hierarchically from cylindrical slab-like layers that consist of the 36 subunits arranged as a hexamer of Hfq hexamers. Although thought-provoking, the physiological meaning of such an Hfq fibrillar structure is unclear as the fibres were produced under less than physiological conditions (5 mM Tris-HCl, pH 8.0, and 5 mM NaCl). Although physiological concentrations of Hfq (5 mM) were used to form these fibres, the presence of other Hfq-binding proteins with higher Hfq-binding affinities, might interfere in their creation. Regardless, these fibres do bind RNA, albeit rather weakly. Perhaps, this fibrillar structure is simply a good storage form of the protein or is involved in nucleoid function. Clearly, any functional relevance of this intriguing form of Hfq will require additional work.
Conclusion Studies on Hfq over the past two years have demonstrated that this Sm protein has at least two major RNA binding sites, a Proximal Site, first identified in X-ray crystallographic studies of the S. aureus Hfq–AU5G complex and a Distal Site, which was discovered through a series of structure-based site-directed mutants, in vivo reporter studies and a variety of RNA binding studies. Additional mutational and structural work will be needed to provide detailed understanding of how sRNAs, mRNAs and poly(A) tails bind these sites. Also, any potential RNA binding role of the basic rim of the E. coli Hfq that connects the Proximal and Distal Sites will need to be clarified by additional biochemical and structural work. We have been presented with conclusive data that as part of its function, Hfq binds to several proteins, including RNase E and PNP, which are involved in mRNA mediated decay, and the ribosomal subunit S1 which, together with Hfq, is needed for Qb replication. Defining the binding mechanism(s) that Hfq utilizes in its interactions with these proteins is the next task. One www.sciencedirect.com
intriguing possibility is that the C-terminal tail of Hfq might be involved, because the tail appears to be dispensable in the RNA binding function of Hfq and is rich in serine, glutamine and acidic residues often present at protein–protein interfaces. Identification of other binding partners will also likely be an area of fruitful pursuit. Other open questions that need to be addressed include where does ATP bind on Hfq and what is the function of its ATPase activity. A final word of caution, just because a given organism has an hfq gene, does not mean that it produces a functional Hfq protein or that this is involved in sRNA function. A case in point is S. aureus: as noted, S. aureus Hfq binds sRNAs tightly in vitro and thus makes an excellent model system for studying the biochemical and structural basis of this more general Hfq function [17]. Moreover, S. aureus Hfq can bind RNAIII, the 514 nucleotide RNA effector molecule of the agr system [55], which is a major global regulator in S. aureus [56]. However, Hfq has no detectable effect on RNAIII–target mRNA (spa mRNA) complex formation, as these RNAs interact rapidly thereby precluding the need for Hfq. Moreover, the hfq gene is transcribed very weakly in multiple strains of this bacterium, most probably because of major deletions in its promoter region, resulting in insignificant amounts of the protein. Finally, an hfq deletion strain has no detectable phenotypic effects of bursa infections, produces wild type levels of a-haemolysin and protein A, which require RNAIII–mRNA interactions, and shows no detectable effect of RNAIII inhibition of the synthesis of Rot, a major pleiotropic transcription factor [57]. Thus, it would appear that in staphylocci, Hfq function has been superseded by RNAIII and hence this riboregulator has been downsized by mutational loss of its major promoter. Perhaps Hfqs of other bacteria have similarly diminished roles.
Acknowledgements This work has been supported by funds provided by the endowment of the Robert A Welch Distinguished University Chair in Chemistry, Grant number G0040. We would like to thank Poul Valentin-Hansen for introducing Hfq to us, Gigi Storz for her helpful suggestions and patience and Maria Schumacher for her comments and early outstanding contributions to our understanding of Hfq structure and function. We apologise to those whose work was not discussed due to space limitations.
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22. Storz G, Opdyke JA, Zhang A: Controlling mRNA stability and translation with small, noncoding RNAs. Curr Opin Microbiol 2004, 7:140-144. 23. Majdalani N, Vanderpool CK, Gottesman S: Bacterial small RNA regulators. Crit Rev Biochem Mol Biol 2005, 40:93-113. 24. Livny J, Waldor MK: Identification of small RNAs in diverse bacterial species. Curr Opin Microbiol 2007, 10:96-101. 25. Gottesman S: The small RNA regulators of Escherichia coli: roles and mechanisms. Annu Rev Microbiol 2004, 58:303-328. 26. Storz G, Altuvia S, Wasserman KM: An abundance of RNA regulators. Annu Rev Biochem 2005, 74:199-217. 27. Geissmann TA, Touati D: Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J 2004, 23:396-405. 28. Lease RA, Woodson SA: Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA. J Mol Biol 2004, 344:1211-1223. 29. Afonyushkin T, Vecˇerek B, Moll I, Bla¨si U, Kaberdin VR: Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB. Nucleic Acids Res 2005, 33:1678-1689.
10. de Haseth PL, Uhlenbeck OC: Interaction of Escherichia coli host factor protein with oligoriboadenylates. Biochemistry 1980, 19:6138-6146.
30. Hajnsdorf E, Re´gnier P: Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I. Proc Natl Acad Sci USA 2000, 97:1501-1505.
11. de Haseth PL, Uhlenbeck OC: Interaction of Escherichia coli host factor protein with Qb ribonucleic acid. Biochemistry 1980, 19:6146-6151.
31. Mohanty BK, Maples VF, Kushner SR: The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. Mol Microbiol 2004, 54:905-920.
12. Brescia CC, Mikulecky PJ, Feig AL, Sledjeski DD: Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA 2003, 9:33-43.
32. Folichon M, Arluison V, Pellegrini O, Huntzinger E, Re´gnier P, Hajnsdorf E: The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation. Nucleic Acids Res 2003, 31:7302-7310.
13. Moll I, Leitsch D, Steinhauser T, Bla¨si U: RNA chaperone activity of the Sm-like Hfq protein. EMBO Rep 2003, 4:284-289. 14. Mikulecky PJ, Kaw MK, Brescia CC, Takach JC, Sledjeski DD, Feig AL: Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat Struct Mol Biol 2004, 11:1206-1214. 15. Sun X, Wartell RM: Escherichia coli Hfq binds A18 and DsrA Domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites. Biochemistry 2006, 45:4875-4887. This study confirmed the results of Mikulecky et al. [14] and also highlighted the importance of ‘edge on’ residues in RNA binding. Somewhat puzzling is the different binding stoichiometry that these investigators derive versus that derived by Mikulecky and coworkers. 16. Azam TA, Ishihama A: Twelve species of the nucleoidassociated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem 1999, 274:33105-33113.
33. Folichon M, Allemand F, Re´gnier P, Hajnsdorf E: Simulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails. FEBS J 2005, 272:454-463. 34. Vytvytska O, Moll I, Kaberdin VR, von Gabain A, Bla¨si U: Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding. Genes Dev 2000, 14:1109-1118. 35. Valentin-Hansen P, Johansen J, Rasmussen AA: Small RNAs controlling outer membrane porins. Curr Opin Microbiol 2007, 10:152-155. 36. Vecˇerek B, Moll I, Bla¨si U: Translational autocontrol of the Escherichia coli hfq RNA chaperone gene. RNA 2005, 11:976-984. 37. Sonnleitner E, Napetschnig J, Afonyushkin T, Ecker K, Vecˇerek B, Moll I, Kaberdin VR, Bla¨si U: Functional effects of variants of the RNA chaperone Hfq. Biochem Biophys Res Commun 2004, 323:1017-1023.
17. Schumacher MA, Pearson RF, Møller T, Valentin-Hansen P, Brennan RG: Structures of the pleiotropic translational regulator Hfq and an Hfq–RNA complex: a bacterial Sm-like protein. EMBO J 2002, 21:3546-3556.
38. Ziolkowska K, Derreumaux P, Folichon M, Pellegrini O, Re´gnier P, Hajnsdorf E: Hfq variant with altered RNA binding functions. Nucleic Acids Res 2006, 34:709-720.
18. Tsui HCT, Leung HCE, Winkler ME: Characterization of broadly pleiotropic phenotypes caused by an insertion mutation in Escherichia coli K-12. Mol Microbiol 1994, 13:35-49.
39. Sauter C, Basquin J, Suck D: Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res 2003, 31:4091-4098.
19. Brown L, Elliot T: Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires Host Factor I, an RNA-binding protein encoded by the hfq gene. J Bacteriol 1996, 178:3763-3770.
40. Nikulin A, Stolboushkina E, Perederina A, Vassilieva I, Bla¨si U, Moll I, Kachalova G, Yokoyama S, Vassylyev D, Garber M et al.: Structure of Pseudomonas aeruginosa Hfq protein. Acta Crystallogr D Biol Crystallogr 2005, 61:141-146.
20. Muffler A, Fischer D, Hengge-Aronis R: The RNA-binding protein HF-I, known as host factor for phage Qb RNA replication, is essential for rpoS translation in Escherichia coli. Genes Dev 1996, 10:1143-1151.
41. Wilusz CJ, Wilusz J: Eukaryotic Lsm proteins: lessons from bacteria. Nat Struct Mol Biol 2005, 12:1031-1036.
21. Muffler A, Traulsen DD, Fischer D, Lange R, Hengge-Aronis R: The RNA-binding protein HF-I plays a global regulatory role, which is largely, but not exclusively, due to its role in expression of the sigmaS subunit of RNA polymerase in Escherichia coli. J Bacteriol 1997, 179:297-300. Current Opinion in Microbiology 2007, 10:125–133
42. Se´raphin B: Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs. EMBO J 1995, 14:2089-2098. 43. Arluison V, Derreumaux P, Allemand F, Folichon M, Re´gnier P, Hajnsdorf E: Structural modelling of the Sm-like protein Hfq from Escherichia coli. J Mol Biol 2002, 320:705-712. www.sciencedirect.com
Hfq structure, function and ligand binding Brennan and Todd 133
44. Sukhodolets MV, Garges S: Interaction of Escherichia coli RNA polymerase with the ribosomal protein S1 and the Sm-like ATPase Hfq. Biochemistry 2003, 42:8022-8034. 45. Thore S, Mayer C, Sauter C, Weeks S, Suck D: Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA: common features of RNA binding in archaea and eukarya. J Biol Chem 2003, 278:1239-1247. 46. Stark H, Dube P, Lu¨hrmann R, Kastner B: Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle. Nature 2001, 409:539-541. 47. Tolstorukov MY, Virnik KM, Adhya S, Zhurkin VB: A-tract clusters may facilitate DNA packaging in bacterial nucleoid. Nucleic Acids Res 2005, 33:3907-3918. 48. Arluison V, Hohng S, Roy R, Pellegrini O, Gingery M, Re´gnier P, Ha T: Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA. Nucleic Acids Research 2007 doi: 10.1093/nar/gkl1124. The authors used real-time FRET to show that Hfq promotes strandexchange, in which the internal structure of rpoS is replaced by pairing with DsrA as is the reverse reaction, but over a longer time scale. This study should encourage others to utilize FRET to study other Hfq regulated sRNA–mRNA systems. 49. Morita T, Maki K, Aiba H: RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs. Genes Dev 2005, 19:2176-2186. This study demonstrated the direct interaction between Hfq and RNase E and localized the Hfq binding site to the C-terminal scaffold region of the enzyme. Moreover, Hfq binding to RNase E appeared to preclude RNase E binding to enolase and RhlB, other enzymes of the degradosome but not to PNP. 50. Morita T, Mochizuki Y, Aiba H: Translational repression is sufficient for gene silencing by bacterial small non coding RNAs in the absence of mRNA destruction. Proc Natl Acad Sci USA 2006, 103:4858-4863.
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51. Morita T, Kawamoto H, Mizota T, Inada T, Aiba H: Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phophosugar stress in Escherichia coli. Mol Microbiol 2004, 54:1063-1075. 52. Masse´ E, Escorcia FE, Gottesman S: Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 2003, 17:2374-2383. 53. Arluison V, Mura C, Romero Guzma´n M, Liquier J, Pellegrini O, Gingery M, Re´gnier P, Marco S: Three-dimensional structures of fibrillar Sm Proteins: Hfq and other Sm-like proteins. J Mol Biol 2005, 356:86-96. Using biophysical approaches and electron microscopy and image analysis, this study showed that under conditions of higher pH and low ionic strength, Hfq is able to form a fibrillar structure. The functional significance of such a structure is unknown. 54. Mura C, Kozhukhovsky A, Gingery M, Phillips M, Eisenberg D: The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs). Protein Sci 2003, 12:832-847. 55. Huntzinger E, Boisset S, Saveanu C, Benito Y, Geissmann T, Namane A, Lina G, Etienne J, Ehresmann B, Ehresmann C et al.: Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. EMBO J 2005, 24:824-825. 56. Recsei P, Kreiswirth B, O’Reilly M, Schlievert P, Gruss A, Novick R: Regulation of exoprotein gene expression by agr. Mol Gen Genet 1986, 202:58-61. 57. Geisinger E, Adhikari RP, Jin R, Ross HF, Novick RP: Inhibition of rot translation by RNAIII, a key feature of agr function. Mol Microbiol 2006, 61:1038-1048. This study on the noncoding RNA, RNAIII, from S. aureus, reveals that Hfq does not play a role in agr function. Moreover, evidence is presented which questions whether or not Hfq plays any role in sRNA function in S. aureus, especially given its nearly nonexistent protein levels.
Current Opinion in Microbiology 2007, 10:125–133
Current Opinion in Microbiology 2007, 10:125–133 This review comes from a themed issue on Cell regulation Edited by Gisela Storz and Dieter Haas Available online 28th March 2007 1369-5274/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2007.03.015
Introduction Escherichia coli Hfq (host factor required for phage Qb RNA replication, also known as host factor 1) is a 102 amino acid residue (11.2 kDa) protein that was first identified as an endogenous bacterial protein which, along with the ribosomal protein S1, was necessary for the replication of the RNA plus-strand of bacteriophage Qb [1–3]. Subsequent phylogenetic analyses revealed the presence of Hfq orthologues in approximately half of all sequenced Gram-positive and Gram-negative bacteria, with several bacteria such as Bacillus anthracis encoding more than one hfq gene [4,5]. At least one archaeon, Methanococcus jannaschii, contains a protein that is related to Hfq. The Hfq family of proteins are thermostable, range in length from 70 to 110 amino acid residues and form homohexamers [2,5–7]. In E. coli, Hfq is highly abundant with an estimated 50 000 to 60 000 copies (10 000 www.sciencedirect.com
hexamers) per cell, of which 80% to 90% are found in the cytoplasmic fraction in association with ribosomes [8]. Given the role of in the replication of the Qb RNA virus and its association with ribosomes, early studies showed that this protein bound RNA and displayed its highest affinities for poly(A) and single-stranded AU-rich tracts [9–11]. More recently, the latter preference has been refined, whereby Hfq has been shown to bind with greater avidity to RNA sites containing a short, single-stranded stretch of uridines and adenosines, which are either preceded or followed by a stem-loop structure [12– 14,15]. The remaining intracellular Hfq is associated with the nucleoid and was reported to bind intrinsically curved DNA using phased A-tracts [16]. By contrast, Hfq does not bind the shorter single-stranded oligodeoxyribonucleotide dA7 [17]. Beyond Qb replication, the innate importance of Hfq in cellular function became clear when an hfq null mutant was constructed in E. coli [18]. This mutant showed pleiotropic effects, including decreased growth rates, increased sensitivity to ultraviolet light, mutagens and oxidants and increased cell length. Moreover, an hfq mutation activates or suppresses the synthesis of more than 50 proteins, due in part to the requirement of Hfq for the efficient translation of the rpoS gene, which encodes ss, the sigma factor expressed under a variety of stress conditions and in stationary phase [19–21]. One of the first connections between the ability of Hfq to bind small, noncoding RNAs (sRNAs) and its role in post-transcriptional regulation became evident in studies on OxyS, a 109 nucleotide regulator of the oxidative stress response. This sRNA was shown to bind directly to Hfq and thereby increase OxyS RNA interaction with rpoS mRNA as part of its mechanism to inhibit rpoS translation [5,22]. The now-observed wide-spread use of Hfq in RNA metabolic processes is underscored by the finding that 22 sRNAs of E. coli bind Hfq, a probable requirement in order for them to carry out their cellular functions through antisense recognition of target mRNAs [23]. Clearly the involvement of Hfq in post-transcriptional regulation will surely grow as methods to detect novel sRNAs improve [24].
Hfq as a post-transcriptional regulator Hfq carries out its post transcriptional regulatory function of several genes by binding trans-encoded sRNAs and perhaps their mRNA targets in order to facilitate their direct interaction. Hence, Hfq is a riboregulator and, ever since these early studies, it has been shown to interact functionally with over a dozen sRNAs (the subject of many of the reviews in this issue) [22,23,25,26]. This Current Opinion in Microbiology 2007, 10:125–133
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ultimately enables stabile sRNA–mRNA hybrid duplexes to form through small stretches of complementary sequences. One outcome of such binding is the removal of a translation-inhibitory structure that subsequently enables ribosome access to the transcription initiation region or the creation of an sRNA–mRNA structure that blocks ribosome access to that region. In its riboregulatory role, Hfq has been called an RNA chaperone because nuclease digestion and protection experiments and hydroxy radical footprinting of some sRNAs (e.g. OxyS and Spot 42) and mRNAs (e.g. sodB and ompA) have shown significant protection pattern differences arising from the binding of Hfq indicating that Hfq-binding changes their structures [13,27]. In further support of this designation as a chaperone, Hfq can dissociate or be proteolytically removed from target mRNA or sRNA– mRNA complex, yet the newly formed structures are stabile [13,28,29]. By contrast Hfq does not alter the secondary structure of DsrA, at least not significantly, but more probably changes the tertiary structure and brings about sRNA–mRNA base pairing by increasing the local concentration of the regulatory RNA and its target [12]. Regardless, both mechanisms (active reconfiguration of structure or simple concentration of sRNA– mRNA targets) would bring about the formation of the sRNA–mRNA complex. In addition to regulating translation in an sRNAdependent manner, Hfq modulates the decay of some mRNAs, (e.g. rpsO, which encodes for ribosomal protein S15) by binding to their poly(A) tails, stimulating poly(A) adenylation by poly(A) polymerase I (PAP I) and protecting this message from polynucleotide phosphorylase (PNP), RNase II and RNase E, enzymes involved in mRNA degradation [30–33]. Hfq has also been shown to modulate by two distinct mechanisms the half-life of ompA mRNA, which encodes the major outer membrane protein of E. coli [34,35]. Perhaps not unexpectedly, Hfq also has been found to autoregulate its own expression at the translational level by binding two sites on the hfq message thereby inhibiting the formation of the translational initiation complex [36]. The goal of this opinion piece is to briefly review the known structures of Hfq, to focus on the latest Hfq–RNA binding studies and, in combination with some older studies, to propose a structural, albeit speculative, model for Hfq–poly(A) binding, and to describe results revealing interactions between Hfq and components of the degradosome, as well as Hfq fibre formation.
Hfq is an Sm Protein Perhaps one of the largest remaining hurdles in fully understanding Hfq function is the lack of relevant high-resolution structures. Regardless, the four described Hfq structures — three apoproteins and one with bound RNA — have provided invaluable insight into the RNA Current Opinion in Microbiology 2007, 10:125–133
binding mechanisms of Hfq, either directly or in conjunction with site-directed mutagenesis studies [14,15,37,38]. The apoprotein structures include Hfq from Staphylococcus aureus [17], E. coli (residues 1–72) [39] and most recently Pseudomonas aeruginosa [40]. The structure of the only Hfq–RNA complex is that of S. aureus Hfq bound to the hepta-oligoribonucleotide, AU5G [17]. All structures revealed unequivocally that Hfq is an Sm protein. Sm and Sm-like proteins are found in eukaryotes and archaea and are involved in multiple aspects of RNA metabolism including splicing and mRNA decay [41]. These proteins are characterized by two highly conserved regions, the so-named Sm1 and Sm2 motifs [42]. The early difficulty in assigning Hfq to the Sm family was the lack of sequence homology in the Sm2 motif. However, by 2002 several groups had correctly included Hfq proteins in the greater Sm and Smlike superfamily [4,6,7,43]. Hfq forms a toroid with an outer diameter of 70 A˚ and a ‘thickness’ of 25 A˚. The central pore is 8 A˚ to 12 A˚ wide in the apoprotein structure. The structures of all Hfq proteins are characterized by an N-terminal a helix followed by five b strands that form a highly bent sheet that display the topology b5a1b1b2b3b4 (Figure 1). The Sm1 motif encompasses the first three b strands, whereas the Sm2 motif is composed of b strands 4 and 5. The cyclic hexamer is formed primarily by interactions between residues from b4 and b5 from the apposing subunit. The lone a helix sits on top of the sheet on what is now referred to as the ‘distal side’. The subunit structures of Hfq and Sm proteins are very similar and display root mean squared deviations ranging from 0.37 A˚2 to 0.52 A˚2 (Hfq to Hfq comparison) and 0.85 A˚2 to 1.3 A˚2 (Hfq to archaeal Lsm and Human Sm comparison) [39]. Thus, whether these proteins are hexamers or heptamers impinges little on the structures of their individual subunits. Intriguingly Hfq has been reported to have an ATPase activity, albeit relatively weak [44]. Sequence comparison between Hfq and the heat shock protein, ClpB, revealed some homology between the ATP binding site of ClpB and a stretch of Hfq sequence that includes b2 (Figure 1). This ‘modified Walker A box’ is rather open, and how and where ATP binds, and what is the role of the ATPase activity are under investigation.
Hfq binds sRNA and AU-rich sequences on the proximal side The structure of the S. aureus Hfq in complex with the hepta-oligoribonucleotide, AU5G, has provided initial information on how uridine-rich stretches bind, as well as information on one mechanism of adenosine binding (Figure 2) [17]. The major contributors to RNA binding are located in the Sm1 and Sm2 motifs with residues on the loops between b2 and b3 and b4 and b5 playing a central role. The RNA expands and fills the central, basic pore on the ‘proximal side’ of the hexamer and binds in a www.sciencedirect.com
Hfq structure, function and ligand binding Brennan and Todd 127
Figure 1
Overlay of the structures of four bacterial Hfq proteins. The Hfq structures of P. aeruginosa (PDB code 1U1T), E. coli (PDB code 1HK9), and S. aureus with (PDB code 1KQ2) and without RNA (PDB code 1KQ1) are depicted as ribbons and colored cyan, salmon, yellow and magenta, respectively. The view is into the ‘Proximal’ face. The bound RNA, AU5G, is shown as solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored white, blue, red and orange, respectively. The dashed line envelopes one monomer of the hexameric protein with the b sheets colored magenta and the single a helix in cyan. The secondary structures are labeled for this monomer, as are the b strands b40 and b50 , which form the majority of the inter-subunit interface. The ‘modified Walker A box’, which is proposed to be involved in ATP binding and would encompass strand b2 [44], is labeled on one subunit and shown in blue.
circular manner except for the 30 guanosine, which is located at the entrance of the pore (Figures 1 and 3a). Each of the six potential AU nucleotide binding pockets is composed of residues from two adjacent subunits. Both uridine and adenosine bind similarly, whereby their nucleobases stack against the aromatic side chain of residue Tyr42 and the same residue from a neighboring subunit (Figure 2). This residue is a phenylalanine in the Hfqs of E. coli and P. aeruginosa and identical base-side chain stacking interactions are expected. Other residues involved in binding include Sm1 residue Lys41, which uses its main-chain carbonyl oxygen atom to interact with N1 and N9 atoms of the uracil and adenine rings, respectively, as well as its side-chain nitrogen to hydrogen bond to the O4 oxygens of some uracils. The methylene side chain of residue Lys41 also stacks with some uracils. Sm2 residue Lys57, which is part of the highly conserved KH motif found in all Hfq proteins, makes a hydrogen bond to the uracil O2 atom, whilst Sm2 residue His58 interacts www.sciencedirect.com
Figure 2
Hfq–RNA interactions in the Proximal Site. These contacts are observed in the S. aureus Hfq–AU5G crystal structure (1KQ1). The cyan and yellow coloring of residues and ribbon indicates one of two neighboring subunits that form the binding pocket for each nucleotide. Residues are numbered using the S. aureus Hfq protein sequence. (a) Uridine interactions. The repeating pUp unit is emphasized as solid sticks. (b) Adenosine interactions. The terminal Ap is emphasized by solid sticks. The side chain nitrogen and oxygen atoms are colored blue and red, respectively. The nucleotide nitrogen, oxygen, carbon and phosphorus atoms are colored blue, red, grey and orange, respectively.
with the phosphate group and the O20 of some ribose rings. The latter interaction could be used to favor RNA binding over DNA binding. Unexpectedly, residue Gln8, which is outside the Sm motifs and located on helix a1, is also involved. The side chain of this residue ‘reads’ the uracil and adenine bases and with residues Lys41 and Lys57 is involved in discrimination against cytidine and guanosine binding. The S. aureus Hfq-AU5G structure is informative for certain key aspects of RNA binding, including how short tracts of AU-rich single-stranded RNA can interact with this riboregulator. Of course, one structure does not tell the entire story, and several points must be made. First, given that most Hfq binding sites on their target sRNAs contain stretches shorter than six single-stranded uridines or adenosines that are often interrupted by cytidines and guanosines, it is unlikely that all six binding pockets on the proximal side (or face) are filled simultaneously by a single RNA. Second, these pockets are clearly not the only RNA binding sites on this face as work by the groups of Sledjeski, Feig and Wartell [12,14,15] has demonstrated that other residues of the proximal face, at least in E. coli, as well as other elements of sRNAs, that is their stems and loops, are involved in binding. Interestingly, one of these studies also indicated that residues on the distal face contributed to rpoS mRNA binding [14]. Furthermore, hfq point mutations at positions 3 and 17, which precede the Sm1 motif, cannot support Qb replication, whereas those at Current Opinion in Microbiology 2007, 10:125–133
128 Cell regulation
Figure 3
Electrostatic potential energy surfaces of the known and proposed RNA binding sites of the S. aureus and E. coli Hfq proteins. (a–c) Views of the proximal side and distal electrostatic surfaces of S. aureus Hfq, respectively. (d–f) Views of the proximal, side and distal electrostatic surfaces of E. coli Hfq, respectively (blue is electropositive and red is electronegative). The side view of E. coli Hfq includes a plausible RNA binding cleft that would enable A27 to bind to both the Proximal and Distal Sites. The view of the Distal Site of the E. coli Hfq shows a possible binding site for A18 (i.e. a poly(A) tail). The bound RNAs are shown as solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored white, blue, red and orange, respectively. The electrostatic potential energy surfaces were created by PyMol and the APBS plug-in (Delano Scientific LLC, Palo Alto, CA).
residue 56, (Lys57 in S. aureus; Figure 2) can, implying Qb-binding does not use at least some highly conserved residues of the proximal pore to bind [37]. Intriguingly, a second, ‘external’ RNA binding site, which used residues of the N-terminal a helix and aromatic residues of b2, was observed in the crystal structure of the archaeaon Pyrococcus abyssi Sm core, bound to RNA [45]. Third, the Hfq–AU5G structure suggests that single-stranded RNA might be able to traverse the central pore of the proximal face to reach the Current Opinion in Microbiology 2007, 10:125–133
distal face (Figure 1). In support, the pore has a positive electrostatic surface and, upon RNA binding, has expanded to 15 A˚ wide, which is wide enough to thread a single strand through to the other side (Figures 1 and 3a,d). A 10 A˚ resolution structure of the U1-snRNP indicates RNA does pass through the hole of the Sm heptamer [46]. However, the assembly processes of the heptameric Sm proteins and the hexameric Hfq proteins are quite different. Subsets of Sm subunits assemble on the www.sciencedirect.com
Hfq structure, function and ligand binding Brennan and Todd 129
RNA whereas Hfq proteins are always hexameric. Furthermore, the electrostatic surface of the distal side is fairly negative, which would disfavor threading (Figure 3c,d,f). Of course, RNA threading could require one or more divalent cations, which would neutralize this feature of the pore on the distal face. Clearly more structural, biochemical and in vivo studies will be necessary to clarify these points.
Figure 4
Hfq binds poly(A) tails on the distal side The early studies of de Haseth and Uhlenbeck [10] revealed that the affinity of Hfq for poly(A) RNA displayed a strong length and geometry dependence, in which linear sequences between 15 (pA15) and 27 (pA27) showed extremely tight binding and those of cyclized (pAN) showed higher relative affinity to their linear counterparts when N 18, but lower relative affinity when N 15 [10]. This suggested that the poly(A) binding site on the Hfq hexamer was circular. However, these data could not be reconciled with the AU5G binding site. Recent studies to address the question of the poly(A) binding site on Hfq have provided intriguing results and a likely solution [14,15]. Using poly(A) stretches of either 18 (A18) or 27 (A27), these later studies revealed that residues Tyr25, Ile30 and Lys31 were involved in RNA binding, as their substitution by alanine reduced the affinity of E. coli Hfq for these poly(A)s fivefold to greater than 100-fold. More to the point, these residues are located on the distal side of Hfq, that is, on the side opposite the AU5G binding site. Alanine substitutions of residues Phe42, Tyr55, K56 and H57 on the proximal side, showed only twofold to threefold effects. Furthermore, A27 did not compete with DsrA binding to wild type Hfq, but rather resulted in a supershift. When these data are combined with some RNA stereochemical constraints and the cationic nature of the E. coli Hfq core (residues 1 to 68, pI 10), a plausible, albeit hypothetical, model of poly(A) binding to Hfq can be constructed (Figures 3e,f and 4). First, by contrast to the distal face of the S. aureus Hfq, which does not bind poly(A) with high affinity (TM Link and RG Brennan, unpublished), that of E. coli Hfq is exceedingly positive (Figure 3c,f). Second, examination of the distal face of the E. coli Hfq hexamer reveals that the side chains of residues Tyr25, Ile30 and Leu32 form a hydrophobic pocket (Figure 4). Moreover, the side chain of Lys31 on the adjacent b strand is pointing its positively charged amino group into the solvent, but stacking with the aromatic side chain of Tyr25. Third, the distance between the hydroxyl groups of residue Tyr25 of each subunit is 17 A˚. The distance that a trinucleotide repeat (A3) would span in the C30 endo conformation, which is that favored by RNA, is 17.7 A˚ (3 5.9 A˚). Thus, only a very slight adjustment in the RNA backbone or the Tyr25 side chain would enable one adenine nucleotide of a www.sciencedirect.com
Model of a hypothetical interactions between one adenine of a poly(A) RNA and Hfq residues of the E. coli Distal Site. These residues have been shown to affect poly(A), but not sRNA, polyU or AU5G binding to Hfq. Residues, shown as solid sticks, and secondary structures, shown as ribbons, from apposing subunits that form the putative RNA binding pocket are colored cyan and yellow. Nitrogen and oxygen atoms of each residue are colored blue and red, respectively. The bound poly(A) RNA is shown in solid sticks with carbon, nitrogen, oxygen and phosphorous atoms colored grey, blue, red and orange, respectively.
triplet to use each pocket. Moreover, residues Gln52 and Asn28, which are highly conserved amongst Hfq proteins, are nearby to read the hydrogen bond donors and acceptors of the adenine ring, and residue Lys31 is in position to interact with the phosphate backbone (Figure 4). Coincidentally A18 in the C30 endo conformation would occupy the entire site nearly perfectly (18 nucleotides 5.9 A˚ = 106.2 A˚; the circular distance starting and ending at the hydroxyl oxygen of any Tyr25 subunit is 6 17 A˚ = 102 A˚). Such a binding site might also explain the need for longer poly(A) tracts for highaffinity binding as the Hfq-adenosine interactions provided by individual pockets are probably insufficient until a more cooperative binding mode can be generated by longer A-tracts. Again, this model is quite speculative and ultimately, a structure of Hfq bound to poly(A) will be necessary to provide the details of this protein–RNA interaction. Given the stereochemistry, length and very basic nature of the distal face, one could speculate that this side of Hfq might well bind DNA A-tracts [16], which are found Current Opinion in Microbiology 2007, 10:125–133
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periodically throughout the E. coli chromosome, and thus in the nucleoid, and are associated with intrinsic DNA curvature [47]. Another notable feature of E. coli Hfq is the positive electrostatic surface of the trough that connects the proximal and distal faces, which again is in sharp contrast to the same area on the S. aureus Hfq, which shows a negative electrostatic surface (Figure 3b,e). Residues Arg16 and Arg17 of helix a1, Arg19 and Lys47 are the major contributors to the cationic nature of the trough. Interestingly, residue Arg16 has been implicated in DsrA binding [12,15] and residues Arg17 and Arg19 are located near the external U7 binding site of Lsm1 [45]. How other sRNAs, as well as DsrA and target RNAs use this site on Hfq will require additional studies.
between the sRNA and target mRNA, which results in the exposure of the rpoS ribosome binding site. Strandexchange takes place as a consequence of the rapid Hfq-mediated association of DsrA and rpoS, followed by slow melting of the rpoS stem region and similarly slow annealing of the two RNAs. Interestingly, these studies also revealed that Hfq disrupts preformed DsrA–rpoS complexes, albeit slowly. The physiological role of such disruption by Hfq is unclear, but could be part of the fine-tuning mechanism of sRNA regulation. It should be noted that these chaperoning activities of Hfq are independent of ATP hydrolysis thereby leaving the function of the ATPase activity of Hfq an open question. Similar FRET studies on other Hfq-regulated systems and those that attempt to measure donor–acceptor distances, should yield new insights into Hfq function.
Hfq–RNA stoichiometry
Hfq interacts with other proteins
Three recent studies have reported rather different results regarding just how many Hfq hexamers bind to one DsrA sRNA, one rpoS mRNA or one A18 molecule. In one study, isothermal titration calorimetric experiments revealed 1:1 (DsrA:Hfq hexamer) and (rpoS:Hfq hexamer) binding stoichiometries but a 2:1 (A18:Hfq hexamer) where A18 binds two identical sites [14]. The 1:1 rpoS: Hfq hexamer stoichiometry found by Mikulecky et al. [14] differs from the 1:2 (rpoS:Hfq hexamer) stoichiometry reported by Lease and Woodson [28], who determined this parameter by gel shift assays. Gel-shift experiments performed by Sun and Wartell [15] showed a 1:2 (DsrA:Hfq hexamer) stoichiometry, similar to that reported by Lease and Woodson. By contrast, fluorescence anisotropy studies on the Hfq binding to A18 show the 2:1 (A18:Hfq hexamer) stoichiometry, which appears to reflect two separate, but nearly equal, Hfq binding events on this poly(A) [15]. The reported differences are somewhat baffling, but could be the result of several factors, such as the different protein purification protocols, and hence their different specific activities, experimental methodologies and conditions, and the purity and homogeneity of their RNA samples. Structural data would be helpful in reconciling these data. In addition to crystallographic studies, single-particle image reconstruction could be a good avenue of pursuit. It would also be interesting to see how Hfq binding to any of its other protein partners influences stoichiometry. Ultimately, we might find that the in vivo stoichiometry is not fixed but rather flexible depending upon the cellular environment.
Given the riboregulatory functions of Hfq, it would be surprising if Hfq did not interact with components of the ribosome, degradosome or other cellular machines that are involved in RNA metabolism. Indeed, S1 has been shown to mediate Hfq binding to RNA polymerase and thereby modulate the transcriptional activity of the enzyme [44]. However, the demonstration of a direct protein–protein interaction between S1 and Hfq awaits. In a more recent study on the role of Hfq in polyadenylation-dependent mRNA decay, Kushner and colleagues [31] found that Hfq copurified with PNP and PAP I, two components of the degradosome, in an sRNA-independent manner. Further, that investigation revealed that Hfq could form a complex with PNP separately. Whether Hfq interacts directly with PAP I alone was not shown. Regardless, an Hfq–PNP–PAP I complex has clear physiological implications for the role of Hfq in regulating the polyadenylation of full length mRNAs that contain Rhoindependent transcription terminators. Hfq could destabilize the terminator stems, which are then direct targets of PAP I activity and ultimately subject to degradation by PNP and RNase II.
Hfq is a chaperone In studies that examined the chaperone activity of Hfq, Ha and colleagues [48] used real-time fluorescence resonance energy transfer (FRET) to monitor the effect of Hfq on promoting intermolecular base pairing between DsrA and rpoS. These studies revealed that Hfq accelerates strand exchange and subsequent annealing Current Opinion in Microbiology 2007, 10:125–133
In studies on the role of RNase E in the degradation of target mRNAs of the sRNAs, SgrS and RhyB, Aiba and coworkers [49,50] found that Hfq together with SgrS and RhyB copurifed with RNase E to form specialized ribonucleoprotein complexes that were distinct from the degradosome, that is PNP and RhlB RNA helicase, were not present. Enolase, another component of the degradosome that plays a crucial role in the rapid decay of ptsG mRNA, the target of SgrS, was also not present [51]. This work identified the C-terminal scaffold domain of RNase E as the Hfq-binding site. This domain had been found previously to participate in RhyB-mediated degradation of target mRNAs [52]. Binding of sRNA to Hfq does not appear necessary for the formation of a stabile Hfq– RNase E complex. Moreover, there is competition between Hfq and the degradosome components, except www.sciencedirect.com
Hfq structure, function and ligand binding Brennan and Todd 131
enolase, for binding to the C-terminal scaffold of RNase E, implying overlapping binding sites. Ultimately, the RNase E–Hfq–sRNA complex results in translational repression and rapid target mRNA degradation. Interestingly, in the absence of RNase E, Hfq binding to these sRNAs still results in translational repression, which appears to be sufficient for the gradual destabilization and destruction of the target mRNAs [50]. One other protein has been shown unequivocally, at least in vitro, to interact with Hfq. This is Hfq itself [53]. Using biophysical approaches and electron microscopy and image analysis at a resolution of 23 A˚, Hfq was shown to form well-ordered fibres that resemble those seen previously in archaeal Sm proteins (SmAPs) [54]. The helical pitch of the Hfq fibrillar structure is 245 A˚ and the corresponding vertical spacing between consecutive helical subunits is 40 A˚. These parameters result in 36 Hfq subunits per helical pitch. The fibres differ in detail with those of SmAPs, forming polar tubes and those of Hfq assembling hierarchically from cylindrical slab-like layers that consist of the 36 subunits arranged as a hexamer of Hfq hexamers. Although thought-provoking, the physiological meaning of such an Hfq fibrillar structure is unclear as the fibres were produced under less than physiological conditions (5 mM Tris-HCl, pH 8.0, and 5 mM NaCl). Although physiological concentrations of Hfq (5 mM) were used to form these fibres, the presence of other Hfq-binding proteins with higher Hfq-binding affinities, might interfere in their creation. Regardless, these fibres do bind RNA, albeit rather weakly. Perhaps, this fibrillar structure is simply a good storage form of the protein or is involved in nucleoid function. Clearly, any functional relevance of this intriguing form of Hfq will require additional work.
Conclusion Studies on Hfq over the past two years have demonstrated that this Sm protein has at least two major RNA binding sites, a Proximal Site, first identified in X-ray crystallographic studies of the S. aureus Hfq–AU5G complex and a Distal Site, which was discovered through a series of structure-based site-directed mutants, in vivo reporter studies and a variety of RNA binding studies. Additional mutational and structural work will be needed to provide detailed understanding of how sRNAs, mRNAs and poly(A) tails bind these sites. Also, any potential RNA binding role of the basic rim of the E. coli Hfq that connects the Proximal and Distal Sites will need to be clarified by additional biochemical and structural work. We have been presented with conclusive data that as part of its function, Hfq binds to several proteins, including RNase E and PNP, which are involved in mRNA mediated decay, and the ribosomal subunit S1 which, together with Hfq, is needed for Qb replication. Defining the binding mechanism(s) that Hfq utilizes in its interactions with these proteins is the next task. One www.sciencedirect.com
intriguing possibility is that the C-terminal tail of Hfq might be involved, because the tail appears to be dispensable in the RNA binding function of Hfq and is rich in serine, glutamine and acidic residues often present at protein–protein interfaces. Identification of other binding partners will also likely be an area of fruitful pursuit. Other open questions that need to be addressed include where does ATP bind on Hfq and what is the function of its ATPase activity. A final word of caution, just because a given organism has an hfq gene, does not mean that it produces a functional Hfq protein or that this is involved in sRNA function. A case in point is S. aureus: as noted, S. aureus Hfq binds sRNAs tightly in vitro and thus makes an excellent model system for studying the biochemical and structural basis of this more general Hfq function [17]. Moreover, S. aureus Hfq can bind RNAIII, the 514 nucleotide RNA effector molecule of the agr system [55], which is a major global regulator in S. aureus [56]. However, Hfq has no detectable effect on RNAIII–target mRNA (spa mRNA) complex formation, as these RNAs interact rapidly thereby precluding the need for Hfq. Moreover, the hfq gene is transcribed very weakly in multiple strains of this bacterium, most probably because of major deletions in its promoter region, resulting in insignificant amounts of the protein. Finally, an hfq deletion strain has no detectable phenotypic effects of bursa infections, produces wild type levels of a-haemolysin and protein A, which require RNAIII–mRNA interactions, and shows no detectable effect of RNAIII inhibition of the synthesis of Rot, a major pleiotropic transcription factor [57]. Thus, it would appear that in staphylocci, Hfq function has been superseded by RNAIII and hence this riboregulator has been downsized by mutational loss of its major promoter. Perhaps Hfqs of other bacteria have similarly diminished roles.
Acknowledgements This work has been supported by funds provided by the endowment of the Robert A Welch Distinguished University Chair in Chemistry, Grant number G0040. We would like to thank Poul Valentin-Hansen for introducing Hfq to us, Gigi Storz for her helpful suggestions and patience and Maria Schumacher for her comments and early outstanding contributions to our understanding of Hfq structure and function. We apologise to those whose work was not discussed due to space limitations.
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