Cycling of the Sm-like Protein Hfq on the DsrA Small Regulatory RNA

doi:10.1016/j.jmb.2004.10.006

J. Mol. Biol. (2004) 344, 1211–1223

Cycling of the Sm-like Protein Hfq on the DsrA Small Regulatory RNA Richard A. Lease and Sarah A. Woodson* T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St. Baltimore, MD 21218-2865 USA

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA–RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5 0 leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA$rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA$rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA$rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA$mRNA complex. q 2004 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: anti-sense RNA; RNA chaperone; non-coding RNA; RNA– protein interactions; riboregulation

Introduction RNA regulation is a widespread mechanism for post-transcriptional control of gene expression. Recently, it has been recognized that bacterial responses to environmental cues such as oxidative stress, osmotic shock and temperature are coordinated through non-coding, small RNAs (sRNAs).1 Many sRNAs base-pair directly with their target mRNAs.2,3 Interactions with sRNAs can alter the translational activity of an mRNA by triggering a conformational change in a translational operator, or may target the mRNA for rapid degradation.4 Hence, bacterial sRNAs are functionally analogous to microRNAs that repress translation or accelerate turnover of eukyarotic mRNAs.5,6 DsrA is an 87 nt sRNA (Figure 1) that increases the translation of rpoS at low temperatures.7 rpoS encodes the stationary phase sS, a pleiotropic Abbreviations used: sRNA, small RNA. E-mail address of the corresponding author: [email protected]

regulator that is an important component of the bacterial stress response circuitry.8,9 In the absence of stimulatory factors, translation of rpoS is repressed by a large stem-loop upstream of the AUG start codon that sequesters the Shine–Dalgarno sequence (Figure 1).10 DsrA is complementary to sequences in the 5 0 leader of rpoS mRNA, and base-pairing between DsrA and rpoS RNA is proposed to disrupt the inhibitory stem-loop, making the ribosome binding site accessible.11 This interaction requires unfolding of secondary structures in both DsrA and rpoS RNA, raising the possibility that proteins are required to mediate formation of the RNA complex in vivo. DsrA also down-regulates expression of hns and other mRNAs,12 thereby effecting a global response to environmental changes.13 Expression of rpoS requires the protein Hfq,14,15 and genetic experiments demonstrated that regulation of rpoS by OxyS, RprA and DsrA sRNAs also requires Hfq.16–18 Hfq was first identified as a host factor for replication of phage Qb19 and is thought to support replication by remodeling secondary

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

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Hfq Release from sRNA–mRNA Complex

to eukaryotic and archaeal Sm proteins, these results may be relevant to the function of Sm complexes in other organisms.

Results Hfq binding sites on DsrA and rpoS

Figure 1. DsrA is complementary to the rpoS mRNA. DsrA sRNA base-pairs with two regions of rpoS mRNA leader (shaded rectangles). This interaction opens an inhibitory stem-loop in the rpoS leader and exposes the Shine–Dalgarno (SD) ribosome binding site.9 The protein Hfq is genetically required for rpoS regulation by sRNAs.

structure or stabilizing long-range interactions in the phage RNA.20,21 Hfq binds at least 20 bacterial sRNAs,22 and has been shown to enhance their interactions with target mRNAs.23–25 Hfq is proposed to facilitate base-pairing between sRNAs and their targets either by altering the secondary structure of one or both partners, or by holding complementary RNAs in close proximity through protein–protein interactions.4,8 How Hfq structurally mediates sRNA interactions is unknown. Electron microscopy, crystallographic and analytical centrifugation studies showed that Hfq forms a hexameric ring of identical 11.4 kDa subunits, and is structurally and evolutionarily homologous to the eukaryotic Sm and Lsm proteins that are involved in pre-mRNA splicing, and mRNA stability and translation.23,24,26–29 Hfq preferentially binds AU-rich RNAs.30,31 In a cocrystal, the oligonucleotide 5 0 AUUUUUG was observed to bind along the internal surface of the hexameric ring in an extended conformation.27 Stem-loops adjacent to unpaired U bases contribute to Hfq interactions with DsrA and OxyS sRNAs,23,32 in a manner reminiscent of Sm binding sites.33,34 We present a thermodynamic and kinetic framework for interactions of Hfq with DsrA sRNA and rpoS mRNA. Nuclease footprinting and gel mobility-shift experiments show that base-pairing between DsrA and rpoS eliminates a strong Hfq binding site in DsrA, resulting in release of Hfq from the sRNA$mRNA complex at low concentrations of protein. By contrast, high concentrations of Hfq trap the RNAs in unreactive complexes. Thus, optimal hybridization is achieved when Hfq occupies one binding site on DsrA. These results suggest that release of Hfq helps drive association of the sRNA with its target. As Hfq is homologous

DsrA and Hfq are proposed to stimulate rpoS translation by opening an inhibitory secondary structure in the mRNA leader.10–12 To understand how DsrA and Hfq alter the structure of rpoS mRNA, the conformations of DsrA and a 140 nt RNA containing the rpoS translational operator were mapped by partial digestion with RNases T1 and T2 (Figure 2A and B). The protection patterns of the individual RNAs were consistent with the secondary structure model of DsrA determined previously by nuclease footprinting (Figure 2C),35 and with the secondary structure model for the rpoS leader based on genetic experiments in Salmonella typhimurium (Figure 2D).10 Core sequences in the rpoS translational operator are conserved between Escherichia coli and related bacteria (Figure 2D, shaded), and could be folded into similar secondary structures.10 Protection of specific sequences from cleavage in the presence of Hfq plus its known binding preferences (about six consecutive unpaired U/A nucleotides) identified the most likely proteinbinding sites on DsrA and rpoS. Footprinting experiments were carried out in conditions in which specific RNP complexes are formed (see below). In DsrA, the U-rich linker between stemloops (SL) I and II (nucleotides 23–35) was protected from cleavage by RNase T2 in 600 nM Hfq (Figure 2C). We observed slight protection of A11-U13 in SL I and U49-A50 in SL II. Conversely, nuclease cleavage at the base of SL I (G18, 21) and of G46 in SL II was enhanced slightly by Hfq. Together, these results suggest that Hfq preferentially binds the unpaired linker between SL I and II and parts of SL II, consistent with previous work.32 In rpoS RNA, the nuclease footprinting data indicated that Hfq most likely binds two sites upstream and downstream of the region complementary to DsrA. U487-U490 in the loop of P3 (5 0 UUAUUU) were protected by Hfq from cleavage by nuclease (Figure 2B and D). Nucleotides downstream of the AUG codon (569–575) were cleaved more frequently in 600 nM Hfq, suggesting that Hfq binds the 5 0 AUUUUG sequence in the complementary strand (nucleotides 443–448; Figure 2D). Further experiments are needed to assess whether either of these two sites contribute to rpoS regulation. Sequences in J3/4 (nucleotides 500–502) and P4 (nucleotides 531–532) were protected weakly in 6 mM Hfq (Figure 2D), perhaps indicating nonspecific interactions. Together, the results suggested that Hfq recognizes unpaired U-rich sequences in rpoS mRNA, without altering its secondary structure significantly.

Hfq Release from sRNA–mRNA Complex

1213

Figure 2. Ribonuclease footprinting of DsrA and rpoS RNA. Before digestion with RNase T1 or T2, (5 0 -32P)-labeled DsrA (89 nt) or rpoS RNA (140 nt) was incubated with buffer (n), 100 nM complementary RNA (D or R), 600 nM or 6 mM Hfq (H), as indicated above each lane. Lanes T1 and T2, control reactions in urea to obtain sequencing ladders; (–), no treatment with nuclease. Representative sequencing gels are shown for (A) DsrA and (B) rpoS RNA. C–E, Summary of nuclease footprinting experiments on predicted secondary structures for DsrA35 and rpoS mRNA;10 (C) DsrA alone; (D) rpoS mRNA; (E) DsrA$rpoS RNA complex. Positions cleaved in native conditions relative to 6 M urea are indicated

1214 DsrA makes the ribosome binding site accessible To determine whether the ribosome binding site becomes accessible after DsrA binds rpoS mRNA, digestion with nuclease was carried out after annealing the two RNAs at 37 8C (Figure 2A and B; lanes D and R). As shown below, these conditions produce a stable DsrA$rpoS RNA complex. Changes in the cleavage pattern were consistent with predicted interactions between DsrA and rpoS RNA.11,12 Regions of DsrA that are complementary to rpoS mRNA were protected from digestion by nuclease in the RNA complex, including A11-U14, G21, and C25-U34 (Figure 2E). G19 of DsrA was cleaved more strongly, consistent with unfolding of SL I in the DsrA$rpoS complex. DsrA protected the top strand of rpoS mRNA from digestion by nuclease (G462–465), while cleavage of the bottom strand was enhanced strongly (Figure 2B and E). These results provide direct evidence that DsrA disrupts the secondary structure of the leader and makes the ribosome binding site accessible. The nuclease footprinting results suggested that DsrA reorganized the entire structure of the rpoS leader. In addition to expected changes near the translation initation site, DsrA enhanced cleavage of nucleotides in P3 and in P2 upstream of the Shine–Dalgarno sequence (Figure 2E). By contrast, Hfq made little further difference to the conformation of rpoS RNA in the presence of DsrA. When 6 mM Hfq was added to the DsrA$rpoS RNA complex, the same sequences were protected as in DsrA and rpoS RNA alone. Exceptions were G46 in DsrA SL 2 and the Shine–Dalgarno site in rpoS RNA, which remained accessible (asterisks; Figure 2C–E). No additional Hfq protection was observed on either RNA in the complex. These results suggested that the conformational changes that occur when DsrA and rpoS base-pair with each other diminish binding of the RNA duplex by Hfq. DsrA and Hfq associate rapidly To understand how Hfq interacts with DsrA and rpoS RNA, we measured the stability of the RNP complexes using native gel mobility-shifts.17 DsrA RNA was labeled at the 5 0 -end with 32P and incubated with 600 nM Hfq at room temperature. Within 30 seconds, all of the DsrA RNA shifted to a band with lower mobility on a native 6% polyacrylamide gel (Figure 3A, D$H I). No further change in the mobility of the complex was observed over two hours at temperatures ranging from 8 8C to 42 8C (data not shown). To estimate the dissociation rate of Hfq, poly(U)

Hfq Release from sRNA–mRNA Complex

RNA was used to trap Hfq released from the DsrA$Hfq complex. Upon addition of excess poly(U) to pre-formed complexes, all of the 32 P-labeled DsrA dissociated from Hfq within 30 seconds (Figure 3A, right). Thus, DsrA$Hfq complexes equilibrate rapidly, although the RNPs are sufficiently stable to be resolved in the gel. Assuming that 95% of complexes dissociate within 30 seconds, and that Hfq binds as a hexamer, the association rate constant is R5!106 MK1 sK2. DsrA contains a strong Hfq binding site Previous work32 and the footprinting experiments described above showed that Hfq recognizes the unpaired U nucleotides adjacent to stem-loop II in DsrA. Incubation of trace amounts of 32P endlabeled DsrA with various concentrations of Hfq yielded two discrete DsrA$Hfq RNPs (D$H I and D$H II; Figure 3B). The difference in mobility suggested a higher stoichiometry of bound Hfq per RNA in the second complex. Excess DsrA favored RNP I over RNP II, consistent with each complex involving only one molecule of DsrA (data not shown). The equilibrium dissociation constants for each RNP were determined by fitting the fraction of bound DsrA versus Hfq concentration to a partition function for Hfq bound to two independent sites on DsrA (Figure 3C; and see Materials and Methods). This yielded dissociation constants (Kd ) of 220(G14) nM and 4.0(G0.3) mM Hfq monomer, respectively (Table 1). The disappearance of DsrA$Hfq RNP I and appearance of RNP II with increasing Hfq could be accounted for only by assuming cooperative association of Hfq with each site, with an apparent Hill coefficient of 2.4. This cooperativity may reflect interactions between Hfq monomers or between hexamers bound at a single site. Titration of DsrA with Hfq at concentrations above the Kd for the first complex (400 or 800 nM DsrA) indicated a stoichiometry of 12 : 1 for RNP I (Figure 3D). This implies the cooperative binding of two Hfq hexamers to DsrA with an apparent Kd of 18 nM Hfq12. Similar experiments suggested a higher stoichiometry for RNP II. Hfq binds rpoS mRNA leader It is not clear whether the requirement for Hfq in rpoS expression is entirely attributable to sRNAs.8,9 Hfq may also affect rpoS translation by interacting with rpoS mRNA. Hfq has been shown to interact directly with ompA and sodB mRNAs.25,36 Gel mobility-shift experiments with the 140 nt rpoS RNA yielded four species of lower mobility

with open triangles (RNase T1) or filled triangles (RNase T2). Weak effects are indicated with a bar. Change in cleavage intensity upon binding of DsrA (D), RpoS (R), 600 nM Hfq (H) or 6 mM Hfq (HH) is indicated by black letters on light circle (protected) or white letters on a dark background (enhanced). The Shine–Dalgarno sequence and rpoS start codon (C1) are in boldface. Shaded sequences in D are invariant between E. coli and related bacteria (see Materials and Methods). The rpoS sequence is numbered from the transcription start site in nplD,55 with major pairings indicated by P.

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Hfq Release from sRNA–mRNA Complex

Figure 3. Formation of DsrA–Hfq complex. A, Kinetics of Hfq–RNA interactions. Left, 300 nM Hfq was added to P-labeled DsrA RNA and incubated at 30 8C for the times shown above each lane (0–2.5 minutes) before loading onto a native 6% polyacrylamide gel. Right, 32P-labeled DsrA was preincubated with Hfq, then challenged with 5 mg/ml of poly(U) RNA for the times shown, before native PAGE. Band D, free DsrA, D$H (I) and (II), DsrA$Hfq complexes. B, Association of 4 nM 32P-labeled DsrA with Hfq protein (0–12.5 mM monomer). DsrA and Hfq were incubated for five minutes at room temperature before native PAGE (see Materials and Methods). Bands are labeled as in A. C, Fraction of bound DsrA versus Hfq monomer concentration. Symbols: C, D$H I; :, D$H II; ,, ICII. Data were fit to two independent sites with dissociation constants K1Z220 nM and K2Z4.1 mM, as described by equation (1a)–(1c) in Materials and Methods. D, Stoichiometry of Hfq:DsrA complexes. DsrA RNA (400 nM) was titrated with Hfq protein, and the fractional saturation of D$H RNP I plotted against the ratio of Hfq monomer to DsrA. The slope of the linear segment was 1/11.3. 32

(Figure 4A, bands R$H I–IV). As we observed with DsrA, these RNP complexes form and dissociate within 30 seconds (data not shown). Titration of rpoS RNA with Hfq at high concentration was consistent with 12 : 1 Hfq:RNA stoichiometry for rpoS$Hfq RNP I (data not shown). Importantly, even the strongest sites on rpoS

mRNA bound Hfq fourfold less tightly than DsrA under these conditions (Figure 4B). The data were well fit by models assuming two binding sites, each with KdZ880 nM (73 nM Hfq12), and one or more weak sites with KdZ4 mM, as described in Materials and Methods. These results were consistent with the nuclease footprinting data, which suggested that

Table 1. Equilibrium binding constants for DsrA and Hfq

DsrACHfq rpoS RNACHfq rpoSD63CHfq DsrA$rpoSCHfq DsrACrpoS RNA DsrACrpoS (600 nM Hfq)

K1 (nM)

Sitesa

K2 (mM)

Sites

221 880 – 980 8.7 5.1

1 2 – 2 1 1

4.0 4.0 3.0 2.4

1 1 2 1

K3 (mM)

8.0

Sites

nb

1

2.4 2.2–4.0 2.0 2.4

Parameters were obtained from global fits of gel mobility-shift data for individual complexes to equations (1a)–(4) as described in Materials and Methods. Binding reactions were carried out at room temperature as described in Materials and Methods. Equilibrium dissociation constants for Hfq are expressed as the monomer concentration at half-saturation, and varied G10% between two trials. The precision of dissociation constants for DsrA and rpoS RNA is G50%. a Number of apparent ligand-binding sites (or bound Hfq complexes) with the preceding dissociation constant. b Cooperativity coefficient of binding (n) with respect to the concentration of Hfq monomers. For rpoS RNA, the range of values obtained from fits to the fraction of R$H I, II and III is given.

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Hfq Release from sRNA–mRNA Complex

Figure 5. Hfq binding in ternary complex. Identification of ternary complexes by native gel electrophoresis. Labeled DsrA (asterisk, lanes 1–7) or labeled rpoS RNA (lanes 8–14) were incubated with the complementary RNA and Hfq protein, as summarized above each lane. Lane 2, DsrAC300 nM Hfq; lanes 3–7, 100 nM rpoS RNA; lanes 9–12, 100 nM DsrA; lanes 4, 6, 10 and 13, 1.2 mM Hfq (C); lanes 5, 7, 11 and 14, 6 mM Hfq (‡). In lanes 3–7 and 9– 12, RNAs were annealed prior to the addition of Hfq. Lanes 6, 7 and 12, poly(U) RNA was added to pre-formed ternary complexes to trap dissociated Hfq as in Figure 1A. Bands: D, free DsrA; R, free rpoS; D$H, DsrA$Hfq RNP I; R$H, rpoS$Hfq RNPS I, II and III; D$R and R$D, DsrA$rpoS RNA (doublet); D$R$H, DsrA$rpoS$Hfq ternary complexes. Figure 4. Hfq binding to rpoS mRNA. A, Gel mobilityshift of a 140 nt T7 transcript containing the translational operator from rpoS mRNA. Incubation of 32P-labeled rpoS RNA with 0–12.4 mM Hfq monomer as in Figure 3B revealed four RNPs (R$H I–IV), corresponding to different numbers of Hfq complexes bound to the RNA. B, Fractional saturation of rpoS complexes; C, R$H I; :, R$H II; ,, R$H IIICIV. Data were fit to equation (2), with two stronger binding sites (K1Z880 nM) and one weak site (K2Z4.0 mM).

Hfq binds sites in P1 and P3 preferentially, and interacts weakly with other sequences in rpoS mRNA. A variant of rpoS RNA (rpoSD63), in which 63 nt of internal stem-loops were replaced by a 5 nt linker (Supplemental Figure S1), lacked the U-rich sequences in P1 and P3. This RNA bound Hfq poorly (Table 1), suggesting that Hfq does not normally bind the Shine–Dalgarno sequence or the region of the rpoS leader that is complementary to DsrA. DsrA, rpoS and Hfq form ternary complexes Induction of rpoS translation requires cooperation between DsrA and Hfq, as deletion of dsrA or hfq decreases expression of RpoS.7,14,15,17 Our results (see above) showed that Hfq binds DsrA and rpoS RNAs independently, although DsrA$Hfq RNP I is the most stable complex. We next asked whether Hfq forms a stable ternary complex with DsrA and rpoS

mRNA, and whether the ternary complex enhances base-pairing between these RNAs. Hfq had been shown to enhance interactions of OxyS and Spot42 sRNAs with their target mRNAs, via ternary complexes containing the protein and both RNAs.23,24 Incubation of 32P-labeled DsrA with rpoS RNA at 37 8C produced an RNA complex that could be trapped in non-denaturing gels (Figure 5, lane 3). The DsrA$rpoS RNA complex migrated as a doublet, suggesting that it has two conformations. In the presence of 1.2 mM or 6.0 mM Hfq, DsrA$rpoS RNA complex was shifted to bands of lower mobility containing ternary complexes (Figure 5, bands D$R$H in lanes 4 and 5). Bands with identical gel mobility were observed when rpoS RNA was radiolabeled (lanes 9–11). The mobility of the ternary complexes was distinct from that of ribonucleoproteins (RNPs) containing Hfq and DsrA (D$H) or Hfq and rpoS RNA (R$H; Figure 5). When ternary complexes were challenged with poly(U), the RNAs were released as a heterodimer, indicating that the RNAs remained base-paired in the ternary complex (Figure 5, lanes 6, 7 and 12). Although the RNA heterodimer forms slowly (see below), Hfq binds and dissociates from the ternary complex within 30 seconds (data not shown), as seen previously for other Hfq complexes. Thus, Hfq equilibrates rapidly among the available binding sites, and the hybridized DsrA$rpoS RNA is stable

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Hfq Release from sRNA–mRNA Complex

over many cycles of Hfq binding and release. Incubation of pre-formed DsrA$rpoS dimer with 0–12 mM Hfq before native PAGE produced four ternary complexes, each representing a different number of Hfq multimers bound to DsrA$rpoS RNA (Supplemental Figure S2). Hfq has an affinity for the DsrA$rpoS complex similar to that for rpoS mRNA (K1Z980 nM versus 880 nM Hfq; Table 1). This is consistent with the footprinting data (Figure 2), which showed that Hfq recognizes principally rpoS residues outside of the region complementary to DsrA. However, Hfq binds the DsrA$rpoS complex fourfold less tightly than DsrA alone. Therefore, Hfq is displaced from its high-affinity binding site on DsrA after annealing with rpoS RNA. It is possible that Hfq binds weakly another site on DsrA. However, this result is consistent with our footprinting studies, which showed that the primary Hfq recognition site in DsrA is masked by conformational changes in the sRNA when it anneals to its mRNA target. Stability of DsrA$rpoS RNA heterodimer The extensive complementarity between DsrA and rpoS mRNA should produce a stable hybridized complex.11,12 The binding equilibrium for the two RNAs was measured by native PAGE (Supplemental Figure S3). In the absence of Hfq, the average binding constant was 9(G1) nM for DsrA$rpoS (Table 1). In the presence of 600 nM Hfq, which is sufficient to saturate DsrA$Hfq RNP I, the dissociation constant for the two RNAs was 5(G0.5) nM (Table 1). Thus, Hfq protein is not required for annealing of the two RNAs, and stabilizes the complexes less than twofold. As very little Hfq is bound to the DsrA$rpoS RNA complex at these concentrations of protein, it is not surprising that Hfq does not stabilize the RNA heterodimer significantly. Hfq accelerates RNA association slightly We next determined whether Hfq increases the rate at which DsrA binds the rpoS leader RNA (Figure 6). Hybridization of DsrA and rpoS RNA proceeded slowly at 30 8C, with a half-time of five minutes in the absence of Hfq (kobsZ0.14 minK1; Figure 6A). That we observe two conformations of the DsrA$rpoS RNA complex in non-denaturing gels suggests a kinetic intermediate in which the two RNAs are annealed only partly. The faster moving band predominates at early times and persists longer at low temperatures, while the upper band becomes more intense after prolonged incubation and at higher temperatures. We speculate that the lower band represents a hybridization intermediate, while the upper band corresponds to the fully annealed RNA heterodimer. Addition of 300 nM Hfq monomer increased the rate of RNA association twofold (Figure 6D). At this concentration of Hfq, DsrA transiently formed a ternary complex with rpoS RNA and

Hfq (Figure 6B, band D$R$H), but accumulated over time in the free DsrA$rpoS RNA heterodimer (Figure 6B, band D$R). In the presence of 6 mM Hfq, higher molecular mass ternary complexes persisted over the course of the reaction (Figure 6C). Under these conditions, the rate of RNA dimerization actually decreased to 0.06 minK1 (Figure 6D). The observed rate of RNA binding correlated very well with the amount of DsrA$Hfq RNP I present at the beginning of the reaction, reaching a maximum of 0.35 min K1 between 300 nM and 600 nM Hfq (Figure 6E). Thus, association of Hfq with its highaffinity site in DsrA enhances recognition of rpoS mRNA slightly, but higher molecular mass Hfq complexes with DsrA or rpoS RNA inhibit this interaction. Since both DsrA and rpoS RNA must unfold in order to base-pair with each other, conditions that destabilize RNA secondary structure should accelerate association of the two RNAs. The observed rate of hybridization with rpoS RNA increased with temperature, to an average of 0.58 minK1 at 42 8C (Figure 6F). Still, Hfq enhanced the annealing rate no more than twofold between 8 8C and 42 8C (Figure 6F) and over a wide range of concentrations of RNA (R.L., unpublished results). The modest effect of Hfq on the rate of base-pairing between DsrA and rpoS mRNA raises the possibility that other factors contribute to this interaction in vivo.

Discussion Translational control by DsrA sRNA There are many examples in bacteria in which translation is regulated by the structure of the mRNA upstream of the open reading frame.37,38 Although some mRNAs interact with proteins or even small metabolites, rpoS is among a growing number of genes now known to be regulated via small RNAs.9 Genetic experiments established a model for translational control of rpoS by the small RNA DsrA, in which DsrA opens the inhibitory secondary structure in the rpoS leader by base-pairing with sequences upstream.7,10,11 Our nuclease footprinting results are remarkably congruent with this model, and provide direct evidence that base-pairing of DsrA increases the accessibility of the ribosome binding site in rpoS mRNA. Recently, a similar result was obtained independently by Feig and co-workers (E. Espinosa, P. Mikulecky & A. Feig, personal communication). Interestingly, we find that DsrA induces additional conformational changes throughout the rpoS RNA. Although DsrA is sufficient to open the rpoS translational operator in vitro, this and other regulatory RNA interactions often involve protein co-factors in vivo.2,4 Proteins may be needed to stabilize the RNA complex or drive conformational rearrangements that permit base-pairing between two RNAs. Many bacterial sRNAs bind Hfq, and Hfq is proposed to enhance their recognition of

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Hfq Release from sRNA–mRNA Complex

Figure 6. Kinetics of target recognition by DsrA. Formation of DsrA$rpoS RNA complexes at 30 8C was measured using 32P-labeled DsrA and 100 nM rpoS RNA as described in Materials and Methods. Samples were loaded onto native gels at the times shown (1–120 minutes); gels were run continuously during the experiment. Control reactions with DsrA only, DsrA plus 300 nM or 6 mM Hfq, or pre-annealed DsrA C100 nM rpoS are indicated above the lanes as in Figure 5. Controls on the right of each panel were incubated for two hours. Bands are labeled as in Figure 5. Ternary complexes labeled D$R$H are equivalent to those in Figure S2, lane 12. (A) no Hfq; (B) 300 nM Hfq monomer; (C) 6 mM Hfq monomer. D, Progress curves for association of DsrA and rpoS RNA, fit to first-order rate equations. C, No Hfq, kobsZ 0.14 minK1; :, 300 nM Hfq, kobsZ0.3 minK1; ,, 6 mM Hfq, kobsZ0.06 minK1. E, Rate of RNA hybridization (C) versus Hfq concentration. Error bars indicate the standard deviation from the average of three trials. Long-dash line, fraction of DsrA bound by Hfq (RNP I) calculated from parameters in Table 1; short-dash line, fraction of rpoS bound by Hfq (RNP ICII). F, Arrhenius plot for RNA hybridization, between 8 8C and 42 8C. Two trials at each temperature are shown. Continuous line, no Hfq (EaZ10.7 kcal/mol); broken line, 300 nM Hfq (EaZ12.4 kcal/mol).

target mRNAs.3 Eukaryotic Sm proteins are clearly necessary for snRNP assembly,34 but it is not known whether they actively promote interactions with pre-mRNA substrates. Hfq binding cycle during target recognition As the Sm-like protein Hfq is necessary for rpoS regulation, an important question is how Hfq interacts with rpoS mRNA and DsrA. Our results show that Hfq binds free DsrA more tightly than rpoS RNA or the DsrA$rpoS RNA complex. Thus, Hfq is displaced from its strong binding site in DsrA during hybridization with rpoS mRNA (Figure 7). This idea is supported by nuclease footprinting results (Figure 2), showing that the single-stranded uridine nucleotides that form the Hfq binding site in DsrA become fully base-paired in the DsrA$rpoS RNA complex. As Hfq binds and dissociates much more rapidly than the two RNAs associate with each other, cycling of Hfq from DsrA$rpoS complex to free DsrA is driven by thermodynamic equilibria rather

than the kinetics of protein release. Only if the concentration of free Hfq is greater than 1 mM would the protein bind rpoS mRNA and the DsrA$rpoS RNA complex. Although Hfq is abundant in E. coli,39 the distribution of Hfq among cellular RNAs will depend on its relative affinity for the available binding sites. Our results suggest a working model for the mechanism of rpoS activation by DsrA in the presence of Hfq (Figure 7). At low to moderate concentrations (!1 mM) of protein, Hfq hexamers occupy primarily a high-affinity binding site on DsrA sRNA, because Hfq binds DsrA four times more avidly than rpoS mRNA. The DsrA$Hfq complex associates with the mRNA, transiently forming a ternary complex. As base-pairs form between DsrA and rpoS RNA, the Hfq recognition site in DsrA is sequestered in the duplex, displacing Hfq from the DsrA$rpoS RNA complex. It is interesting to speculate whether release of Hfq from the sRNA$mRNA complex correlates with translation of the target mRNA instead of degradation. The kinetics of RNA hybridization further

1219

Hfq Release from sRNA–mRNA Complex

to stabilize the final structure, it has been proposed to function as an RNA chaperone.23,42 We find that Hfq has only a small effect on the stability of the DsrA$rpoS RNA complex, suggesting that Hfq is not needed to maintain the complex once it is formed. Surprisingly, Hfq accelerated base-pairing between DsrA and rpoS RNA only twofold under the conditions used here. Passive remodeling of RNA structure (not requiring ATP hydrolysis) is typically sensitive to the structure and stability of the substrate, because it depends on thermal fluctuations between alternative RNA conformations.43 Hfq may be relatively ineffective against the stable secondary structure of our rpoS substrate. Because DsrA binds rpoS RNA slowly in our assays (0.09 minK1 or 2!104 MK1 sK1; 20 8C), especially in the low temperatures at which DsrA induces rpoS translation,7,44 other factors may be required to accelerate this RNA interaction in vivo. Recruitment of target mRNAs by Hfq Figure 7. Hfq cycling during association of DsrA and rpoS mRNA. Hfq protein equilibrates rapidly among available RNA-binding sites. Below 1 mM Hfq monomer, Hfq occupies principally a high-affinity site on DsrA sRNA (circle). The DsrA$Hfq complex associates with rpoS mRNA in the closed conformation, forming a ternary complex. Base-pairing with DsrA opens the stem-loop in the rpoS leader, permitting translation of sS. The Hfq binding site on DsrA is masked, causing Hfq to dissociate from the sRNA$mRNA complex. Above 1 mM, Hfq also binds rpoS mRNA. High concentrations of Hfq antagonize hybridization with DsrA.

suggest that Hfq release favors the formation of a stable RNA heterodimer. The rate of DsrA and rpoS mRNA association correlates strongly with the presence of DsrA$Hfq RNP I, suggesting that this RNP makes productive interactions with target mRNAs (Figure 6D). By contrast, the higher concentrations of Hfq needed to saturate binding sites on rpoS RNA inhibit base-pairing with DsrA. One explanation for this inhibition is that Hfq must dissociate from an intermediate form of the DsrA$rpoS complex in order for the two RNAs to hybridize completely. Alternatively, association of Hfq with an internal stem-loop in the rpoS leader could stabilize its secondary structure, antagonizing interactions with DsrA. We note that Hfq interactions with rpoS mRNA in vivo could be affected by proteins40 or rpoS sequences41 not present in our in vitro system. Role of Hfq in sRNA target recognition Hfq has been shown to enhance interactions between OxyS and fhlA mRNA, and between RyhB and sodB mRNA,25 by destabilizing stem-loops in OxyS or sodB mRNA, respectively. Because Hfq promotes refolding of the RNA but is not required

Hfq may alternatively enhance interactions between sRNAs and their targets by binding both RNAs simultaneously, thereby holding the complementary sequences in close proximity.4 This could occur via interactions between two Hfq hexamers, or simultaneous binding of two RNAs by a single hexamer. While Hfq is primarily a homohexamer in solution,19,23–45 ultracentrifugation and crystallographic data suggest that the protein can form dodecameric complexes.27,28 Our titrations indicated a stoichiometry of 12 Hfq monomers per DsrA or rpoS RNA molecule in the lowest molecular mass complexes (RNP I). A stoichiometry of six Hfq monomers per DsrA, however, has been reported from isothermal calorimetry titrations under slightly different conditions.46 On the basis of this result and mutagenesis experiments, the Hfq hexamer is proposed to bind poly(A) RNA at a separate site from U-rich sequences.32,46 Although the main conclusions of this study do not depend on the precise stoichiometry of the complexes, it will be important to establish the structure of the complex by other methods. Other roles for Hfq in sRNA regulation Given the modest effect of Hfq on hybridization of DsrA and rpoS RNA in vitro, it is possible that Hfq plays other roles in DsrA regulation. First, Hfq protects DsrA and other sRNAs against degradation by RNase E in E. coli.17,47,48 Stabilization of sRNAs may explain much of the in vivo requirement for Hfq.1 Second, Hfq may recruit other proteins to the RNA,8 such as Qb replicase,19 the degradosome49 or ATP-dependent RNA helicases. Sm and Lsm proteins collaborate with many different proteins during splicing, transport and translation,50,51 and Hfq may function similarly. The results reported here provide a foundation for

1220 further work on the mechanism of regulation by small RNAs.

Materials and Methods Sequence comparisons The rpoS leader sequences from the Gram-negative organisms below were aligned using BLAST and pileup analysis. Sequences were obtained from Genbank for E. coli strains K-12, DEC1a, DEC12e, and W-2; Salmonella typhi, Salmonella enterica serovar Typhimurium, Salmonella dublin, Shigella flexneri, Erwinia caratovora, Erwinia chrysanthemi, Erwinia amylovora, Enterobacter cloacae, Yersinia pestis, Yersinia enterocolitica and Xenorhabdus nematophilus. Plasmids DNA templates for preparation of DsrA and rpoS RNA (pUCT7DsrA and pUCT7RpoS2) were constructed by PCR amplification of dsrA and rpoS sequences in chromosomal DNA from E. coli M182. Upstream primers contained a phage T7 promoter, and downstream primers contained a restriction site for run-off in vitro transcription (DraI for DsrA, EcoRI for rpoS, HindIII for rpoSD63). For construction of pUCT7RpoSD63, primers were complementary at their 3 0 ends and were extended without a template. PCR products were purified by agarose gel electrophoresis and ligated into the SmaI site of pUC19. The desired clones were screened by blue/white assay and verified by DNA sequencing.

Hfq Release from sRNA–mRNA Complex

which was found to promote dimerization of DsrA (R.L. & S.W., unpublished results). Reactions were incubated for five minutes at room temperature. Hfq titrations with pre-formed DsrA$rpoS complex were prepared as described above, except that 4 nM 32 P-labeled RNA and 10 nM, 20 nM or 100 nM complementary RNA (2 ml of 5! solution of DsrA or rpoS in TE) were incubated for 20 minutes at 37 8C to form the RNA complex before addition of Hfq (five minutes at room temperature). Association of DsrA and rpoS RNA was measured in the same way, except that the concentration of labeled RNA was 0.4 nM. All samples (2 ml) were analyzed by electrophoresis on native 6% (w/v) (acrylamide 29 : 1 (w/w) /bisacrylamide) gels in 0.5! TBE. Gels were run at 400 V at 8–12 8C, dried, and analyzed using a Phosphorimager (Molecular Dynamics). Determination of equilibrium dissociation constants The fraction of 32P-labeled RNA in each RNP was calculated from the counts in each band relative to the total counts in the lane, and fit to partition functions for cooperative binding of Hfq to two to four sites as described below. Data for individual complexes (RNP I–IV) were fit by a non-linear, least squares procedure (KaleidaGraph, Synergy). Values for K1, K2 and K3 are given in Table 1, and typically varied 10–20%. The cooperativity coefficient n ranged from 2 to 4 (see Table 1). The fraction of DsrA in complexes D$H I or D$H II (fDH I and fDH II, respectively) were fit to a partition function for two non-identical independent sites:

Preparation of RNA substrates Plasmid DNA templates (Plasmid Maxi Kit, Qiagen) were digested to completion with DraI, EcoRI or HindIII (New England Biolabs) prior to in vitro transcription (1–2 ml total volume) as described.52 RNA was treated with calf intestinal phosphatase, 5 0 end-labeled with 32P and purified by denaturing gel electrophoresis according to standard methods. RNA eluted from the gel was precipitated with three volumes of 100% ethanol, dried, resuspended in 50 ml of TE (10 mM Tris (pH 7.5), 1 mM EDTA), and stored at K20 8C. Equilibrium gel mobility-shift assays Hfq protein was purified as described.23 The concentration of the protein was determined by measuring absorbance at 280 nm in 5.5 M guanidine–HCl, using 3280Z3840 MK1 cmK1,53 after treatment with RNase A and two rounds of gel-exclusion chromatography. Prior to use, RNAs were annealed separately in TE by heating for one minute at 70 8C and cooling to room temperature over five minutes. Binding reactions between Hfq and DsrA or rpoS RNA were prepared in a 10 ml volume by combining: 1 ml of 40 nM [32P]RNA (w105 cpm; 4 nM final concentration), 2 ml of TE, 2 ml of 5! Hfq protein or Hfq dilution buffer (50 mM Tris–HCl (pH 7.5), 1 mM EDTA, 250 mM NH4Cl, 10% (v/v) glycerol), 1 ml of 1 mg/ml of tRNA in TE, 1 ml of 30% glycerol plus tracking dyes, and 2 ml of 5! buffer (50 mM Tris–HCl (pH 8.0), 250 mM NaCl, 250 mM KCl). Final conditions were 24 mM Tris–HCl (pH 7.4), 0.5 mM EDTA, 50 mM NH4Cl, 50 mM NaCl, 50 mM KCl, 5% glycerol. These are comparable to conditions used previously,23 except for the omission of 10 mM MgCl2,

cpmDH I ð½Hfq
=K1 Þn Z cpmtotal QDH

(1a)

cpmDH II ð½Hfq
2 =K1 K2 Þn Z cpmtotal QDH

(1b)

ƒDH I Z

ƒDH II Z

QDH Z 1 C ð½Hfq
=K1 Þn C ð½Hfq
2 =K1 K2 Þn

(1c)

where [Hfq] is the concentration of Hfq monomers, K1 is the dissociation constant for the first site, K2 is the dissociation constant for binding to the second site, and n is the Hill coefficient. The partition function QDH in equation (1c) is the sum of terms for each bound state of DsrA (free RNA, D$H I and D$H II). Using the same approach, binding of Hfq to rpoS RNA was fit to a partition function QRH for two equivalent strong sites and one weak site: QRH Z 1 C 2ð½Hfq
=K1 Þn C ð½Hfq
2 =K12 Þn C ð½Hfq
3 =K12 K2 Þn

(2)

The counts in RNP III and IV were summed to simplify the analysis. For rpoSD63, the data were fit to a partition function QRDH for two identical sites: QRDH Z 1 C 2ð½Hfq
=K2 Þn C ð½Hfq
2 =LK22 Þn

(3)

The data were best fit by assuming a weak interaction between Hfq multimers bound at each site (LZ0.85). Two identical and two non-identical sites were assumed for Hfq binding to DsrA$rpoS: QDRH Z 1 C 2ð½Hfq
=K1 Þn C ð½Hfq
=K2 Þn C ð½Hfq
2 =K12 Þn C 2ð½Hfq
2 =K1 K2 Þn C ð½Hfq
3 =K12 K2 Þn C ð½Hfq
4 =K12 K2 K3 Þn

(4)

1221

Hfq Release from sRNA–mRNA Complex

The concentration of free Hfq was corrected for the amount of protein bound to DsrA (16 or 96 nM), where: ½Hfq
free Z ½Hfq
tot K 12nDH ½DsrA
and:

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2004.10.006

nDH Z ð½Hfq
=K1 Þn C 2ð½Hfq
2 =K1 K2 Þn =QDH using the parameters in Table 1. For association of DsrA and rpoS RNA, the fraction bound rpoS RNA versus the concentration of DsrA was fit to the equation for a single binding isotherm. The concentration of free DsrA was corrected according to: ½DsrA
free Z ½DsrA
total K fDR ½rpoS
total where [rpoS]totalZ0.4 nM and fDR is the fraction bound. Estimates of equilibrium binding constant were refined iteratively. Kinetics of DsrA and rpoS RNA association Kinetic binding experiments with 4 nM 32P-labeled DsrA and 100 nM rpoS RNA or vice versa were carried out as described above in a volume of 30 ml. Reactions were prepared by spotting a 10! solution of 32P-labeled RNA on the wall of a microcentrifuge tube. Unlabeled complementary RNA was added to the radiolabeled RNA with a pipet, and both RNAs mixed with buffer and protein in the bottom of the tube by gentle trituration. Reactions were at 30 8C except where stated otherwise. Aliquots (2 ml) were removed from the reaction at defined intervals (0–120 minutes) and loaded directly onto a native gel as above. The gel was run continuously during the experiments. The counts in each band were normalized relative to the total counts in the lane, and the fraction of DsrA$rpoS (D$R)Cternary complexes (D$R$H) were plotted versus time. Progress curves were fit to a first-order rate equation, except for reactions carried out at 8 8C, which were fit to a biphasic rate equation with a burst phase of 6–10%. Equilibrium footprint analysis RNA and RNA–protein complexes were prepared as described above (10 ml) and incubated 20 minutes at 37 8C before treatment with 2 ml of 0.6–0.06 unit/ml of RNase T1 (USB) or RNase T2 (Sigma) at room temperature for 30 seconds. Reactions were stopped by addition of 2 ml of 10 mM aurin tricarboxylic acid (Sigma) on ice. An equal volume of formamide/dye mix (90% (v/v) formamide, TBE, 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol) was added to samples before electrophoresis on 6% or 8% polyacrylamide sequencing gels. Sequence ladders were obtained by nuclease digestion under denaturing conditions as described.54

Acknowledgements We thank A. Zhang and G. Storz for reagents, advice and comments on the manuscript. We thank A. Ishihama and U. Bla¨si for Hfq protein, D. Sledjeski and A. Feig for communication of their results before publication, and M. Fried for helpful advice. This work was supported by grants from the NIH (GM46686, GM60809).

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Edited by M. Belfort (Received 6 August 2004; received in revised form 22 September 2004; accepted 6 October 2004)