Hfq

Molecular Cell, Vol. 9, 23–30, January, 2002, Copyright 2002 by Cell Press

Hfq: A Bacterial Sm-like Protein that Mediates RNA-RNA Interaction Thorleif Møller,1 Thomas Franch,1 Peter Højrup,1 Douglas R. Keene,2 Hans Peter Ba¨chinger,2 Richard G. Brennan,2 and Poul Valentin-Hansen1,3 1 Department of Biochemistry and Molecular Biology University of Southern Denmark-Odense Campusvej 55 DK-5230 Odense M Denmark 2 Department of Biochemistry and Molecular Biology Oregon Health Sciences University Portland, Oregon 97201

Summary The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnutshaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs. Introduction The bacterial Hfq protein is a posttranscriptional regulator that interacts with various RNAs. The protein was discovered as an E. coli host factor required for replication of the RNA phage Q␤ (Franze de Fernandez et al., 1968). More recent studies revealed that E. coli Hfq targets mRNAs for degradation and is essential for efficient translation of the rpoS mRNA encoding the ␴s subunit of RNA polymerase (reviewed in Nogueira and Springer, 2000). In addition, Hfq has been shown to interact with several small regulatory RNAs in E. coli, and the protein is required for OxyS-, DsrA-, and RprA RNA regulation of rpoS (Zhang et al., 1998; Majdalani et al., 2001; Sledjeski et al., 2001; Wassarman et al., 2001). The importance of Hfq is further underlined by an interruption of its gene, which affects the expression of many proteins and causes pronounced pleiotropic phenotypes such as decreased growth rate, increased cell length, and sensitivity to UV light (Tsui et al., 1994; Muffler et al., 1997). Despite significant advances having been made in elucidating the physiological role of Hfq, very little is known about its structure and the manner in which it recognizes and interacts with target RNA. Therefore, the precise mechanisms by which Hfq regulation occurs 3

Correspondence: [email protected]

remains unclear. However, several studies indicate that Hfq accomplishes its regulatory activities by altering target RNA structure or by interfering with ribosome binding (Muffler et al., 1996; Nogueira and Springer, 2000; Vytvytska et al., 2000; Sledjeski et al., 2001). Moreover, biochemical studies indicate that Hfq exists as a homopentamer or homohexamer in solution, and the protein has been reported to bind preferentially to A/Urich RNAs (Kajitani et al., 1994; Franze de Fernandez et al., 1972; Senear and Steitz, 1976). Intriguingly, Hfq shares a number of features with eukaryotic Sm and Sm-like (LSm) proteins (e.g., it binds to different RNAs and affects different aspects of RNA metabolism). These proteins form heteromers with one another and bind as heteromeric complexes to various RNAs, primarily recognizing short U-rich stretches (Branlant et al., 1982; Achsel et al., 1999; Salgado-Garrido et al., 1999; Tharun et al., 2000). At least 15 members of this protein family are conserved throughout the eukaryotic kingdoms and assemble in at least three different complexes each containing seven distinct Sm or LSm polypeptides. The Sm proteins assemble on the spliceosomal small nuclear (sn) RNAs forming Sm core RNP complexes, which are essential for stability and biogenesis of the snRNAs. The LSm2–LSm8 proteins can form an RNA-free ring and bind to U6snRNA. The third complex consisting of protein’s LSm1–LSm7 takes part in mRNA degradation and in replication of a positive-stranded RNA virus (reviewed in Pannone and Wolin, 2000; Nagai et al., 2001; Achsel et al., 2001). Members of the Sm/LSm family possess a common bipartite sequence motif, the Sm motif, consisting of two relatively conserved segments (known as the Sm1 and Sm2 motifs) separated by a region of variable length and sequence (Branlant et al., 1982). The Sm motif constitutes an autonomously folded domain composed of an NH2-terminal ␣ helix followed by a strongly bent fivestranded ␤ sheet and is responsible for RNA binding and for protein-protein interactions among the Sm and LSm proteins (Kambach et al., 1999; Urlaub et al., 2001). Importantly, archaeal genomes have recently been shown to encode either a single or a maximum of two small Sm-related proteins (Salgado-Garrido et al., 1999). Biochemical and structural analysis of such three proteins (i.e., AF-Sm1 and AF-Sm2 from Archaeoglobus fulgidus and SmAP from Pyrobaculum aerophilum) revealed properties of oligomerization and RNA binding similar to the Sm proteins (Achsel et al., 2001; Mura et al., 2001). The AF-Sm proteins bind to RNA with a high specificity for short poly-U sequences and assemble on the RNA to form a homoheptamer ring-like structure, which closely resembles the shape of Sm core RNP and LSm complexes. The crystal structures of the AF-Sm1 and SmAP proteins provide the first high-resolution picture of a heptameric ring structure, and a comparison with the structures of Sm protein dimers reveals closely related monomer fold and intersubunit contacts (Kambach et al., 1999; Mura et al., 2001; To¨ro¨ et al., 2001). Moreover, the structural results suggest how specific

Molecular Cell 24

Figure 1. Hfq Is Essential for Spot 42 RNA Stability Exponential grown cultures of strains NU426 (hfq⫹), TX2822 (hfq1), and TX2761 (hfq2) were treated with rifampicin to block new transcription. Samples were taken at the indicated times and total RNA was extracted. Spot 42 RNA levels were analyzed by Northern blot analysis.

binding to a U-rich Sm site occurs in Sm and LSm complexes. The evolutionary conservation of Sm proteins has led to the idea that the Sm protein family evolved from a single ancestor that was present before the archael and eukaryotic kingdoms diverged (Salgado-Garrido et al., 1999; Achsel et al., 2001). If this is the case, one may expect to find Sm-related proteins in the bacterial domain. Here we report that the 11 kDa, heat stable E. coli Hfq protein contains a Sm1 motif, and oligomerizes to form a complex that exhibits, as judged by electron microscopy, a doughnut-shaped ring structure. In vitro Hfq binds specifically to the antisense Spot 42 RNA, and the stability of this sRNA is severely reduced in Hfq null mutants. High-resolution hydroxyl radical footprints of the Hfq-Spot 42 RNA complex indicate that Hfq possesses RNA binding specificity very similar to the Sm proteins. Finally, we show that Hfq strongly cooperates in short intermolecular base pairing between the Spot 42 RNA and its target site in the galactose operon. Results Hfq Is Required for Spot 42 RNA Stability Our studies of the physiological role of E. coli Spot 42 RNA established that the 109 nucleotide RNA functions as a translational repressor to differentially regulate the galactose operon genes (T. Møller et al., submitted). During this work, we observed that strains lacking the Hfq protein contain low levels of Spot 42 RNA. Given that Hfq is important for the stability of another riboregulator, DsrA (Sledjeski et al., 2001), we determined the half-life of Spot 42 RNA in isogenic hfq⫹ (NU426) and hfq null mutant (TX2822; hfq1) strains. Because hfq is part of an operon and the hfq1 allele carries an insertion of the omega cassette, we included strain TX2761 (hfq2) as control for polar effects on downstream genes (Tsui et al., 1994, 1997). This strain carries the omega cassette in the distal part of the hfq gene and encodes a functional COOH-terminally truncated Hfq protein. Decay of Spot 42 RNA was monitored by Northern blot analysis of total RNA from cells that were treated with the transcription inhibitor rifampicin (Figure 1). The experiments revealed that Spot 42 RNA was stable in the hfq⫹ and hfq2 strains (half-life ⬎ 30 min), whereas the RNA was rapidly degraded in the Hfq knockout strain (half-life ⬍ 2 min). Hfq Interacts with Spot 42 RNA The instability of Spot 42 RNA in the hfq null mutant strain suggests a direct physical interaction with Hfq.

Figure 2. Hfq Forms a Complex with Spot 42 RNA (A) Coimmunoprecipitation of Hfq and Spot 42 RNA. Cell extracts of strains NU426 (hfq⫹) and TX2822 (hfq1) were incubated with Hfqspecific antisera, and immunoprecipitated complexes were bound to protein A agarose. Total immunoprecipitated RNA was extracted and examined for the presence of Spot 42 RNA and 6S RNA by Northern blot analysis. Lanes 1 and 4 (fraction I, control), total RNA from cells extracts; lanes 2 and 5 (fraction II, control), RNA extracted from protein A agarose of untreated extracts; lanes 3 and 6 (fraction III), RNA extracted from protein A agarose of extracts incubated with antisera. Mature 6S as well as a precursor 6S with a 5⬘ end extension are detected in fraction I. (B) Gel mobility shift assays of Hfq binding to Spot 42 RNA. Samples containing 5⬘ end-labeled transcript of Spot 42 RNA (4 nM final concentration) and a 500-fold molar excess of tRNA were incubated with increasing amounts of Hfq, in the absence (lanes 1–4) or in the presence of unlabeled Spot 42 RNA (lanes 5–10). Final monomerconcentrations of Hfq were 20 nM (lanes 2, 5, and 8), 100 nM (lanes 3, 6, and 9), and 500 nM (lanes 4, 7, and 10). No protein was added to the binding reactions (lanes 1, 5, and 8).

To test this in vivo, we carried out immunoprecipitation experiments. Cleared lysates of NU426 and TX2822 cells were incubated with Hfq-specific antisera, and total immunoprecipitated RNA was examined for the presence of Spot 42 RNA and, as a specificity control, for the presence of the 6S RNA. The results of Northern blot analysis of precipitated RNA are presented in Figure 2A. Spot 42 RNA was clearly coimmunoprecipitated from the extract of NU426 cells (lane 3), whereas none of this RNA could be detected in precipitated complexes from the Hfq-deficient strain (even after long exposure; lane 6). While 6S RNA was clearly present in the cell extracts (lanes 1 and 4), no 6S RNA could be detected in the precipitated complexes. Taken together, these results indicate that Hfq and Spot 42 RNA form complexes in vivo. To verify this result, we purified Hfq to near homogeneity and tested for its binding to Spot 42 RNA. Samples containing a fixed amount of 5⬘ end-labeled Spot 42 RNA, a 500-fold excess of tRNA, and increasing amounts of Hfq were incubated, and formation of complexes were examined in gel-retardation assays. The results are presented in Figure 2B and show that Hfq and Spot 42 RNA formed a single, retarded complex, and the formation of this complex was strongly challenged by excess, unlabeled Spot 42 RNA (lanes 6–10). We conclude that Hfq binds specifically to Spot 42 RNA, and the protein has a significant affinity for the RNA (KD ⬇ 2 ⫻ 10⫺8 M, assuming that Hfq binds as a hexamer). Hfq Interacts with Small, Single-Stranded A/U-Rich RNA Segments In order to elucidate nucleotides of Spot 42 RNA in contact with Hfq in the nucleoprotein complex, we per-

Hfq: An Sm-Related Protein 25

LSm proteins (Branlant et al., 1982). A sequence alignment of Hfq proteins is presented in Figure 4. Apart from an asparagine residue, the Sm1 sequence motif is conserved among the Hfq proteins (consisting of five highly conserved amino acids embedded in a characteristic pattern of hydrophobic and hydrophilic residues; see Figure 4) (Achsel et al., 1999). However, no obvious sequences that resemble the Sm2 sequence motif seem to be present in the Hfq proteins. Rather, these proteins possess a second highly conserved segment consisting of a tyrosine, a lysine, and a histidine residue that is flanked by similar hydrophobic residues. As the Sm and LSm family members, the Hfq proteins contain COOHterminal regions of variable length and sequence.

Figure 3. Hfq Binds to Single-Stranded U-Rich Sequences of Spot 42 RNA (A) Hydroxyl radical footprints of free and Hfq-bound Spot 42 RNA. Samples containing 5⬘ end-labeled transcript of Spot 42 RNA (5 nM final concentration) were incubated with increasing amounts of Hfq. No protein added, lane 3; 5, 50, 500, and 1500 nM of Hfq were present in the binding reactions, lanes 4–7, respectively. Alkaline hydrolysis ladder, lane 1. RNase T1 cleavage of Spot 42 RNA under denaturing condition (G-specific cleavage), lane 2. Bars mark regions of Spot 42 RNA that are strongly protected in the presence of Hfq. (B) Secondary structure of Spot 42 RNA (T. Møller et al., submitted). Arrows indicate the Sm-like sites protected by Hfq.

formed hydroxyl radical footprinting, which provides a map of the accessibility to solvent of RNA when it binds to protein. Incubations similar to those used in the gelretardation assays were treated with hydroxyl radicals at 0⬚C and analyzed by gel electrophoresis. The probing is illustrated in Figure 3A, and the information derived from the analysis is summarized in Figure 3B. The footprints of the Hfq-RNA complex show three small patches of protection, three to five nucleotides long, in A/U-rich single-stranded regions of Spot 42 RNA (i.e., positions 57–60, 69–74, and 102–105). These results confirm and further develop the findings of previous reports on the RNA recognition specificity of Hfq, and it seems plausible, based on the gel-retardation results, that protection of all three patches is caused by a single hexameric Hfq. Hfq: An Sm-Related Protein In an attempt to obtain structural information about Hfq, we searched sequence databases for proteins with similarities to Hfq using the BLAST algorithm and Wise2 (intelligent algorithms for DNA searches). The searches revealed that the Hfq protein is conserved in a wide range of bacteria and, intriguingly, the NH2-terminal portion of these proteins possesses significant similarities with the conserved Sm1 sequence motif of the Sm and

Hfq Forms a Hexameric Ring-Shaped Structure A hallmark of the Sm and LSm proteins is their oligomerization into ring-shaped structures. We therefore investigated E. coli Hfq to determine whether this protein also forms a circular structure. Hfq was rotary stained with platinum and carbon and then examined by transmission electron microscopy. A representative view of rotary-shadowed molecules is shown in Figure 5. Most of the particles clearly appear as ring-formed complexes with a central cavity. Similar images were obtained with negatively stained E. coli and S. aureus Hfq (data not shown). The diameter of these particles was 60–70 A˚, thus similar in size and shape to the Sm core particles, the AF Sm1-oligo(U) complex, and the RNA-free LSm protein heteromer (Kastner et al., 1990; Achsel et al., 1999, 2001). In order to determine the stoichiometry of the ringshaped structure, we performed cross-linking experiments with E. coli Hfq and analyzed the molecular mass of cross-linked molecules by MALDI-TOF mass spectrometry. The results revealed products with molecular masses corresponding to monomers (11.031 D, theoretical mass 11.035 D) and up to hexamers of Hfq (67.700 D) and, therefore, provide evidence that the proteins form homohexameric ring-like structures (data not shown). Thus, the bacterial Hfq proteins are likely to constitute a structurally specialized subfamily of the Sm proteins. In support of this view, secondary structure analysis by circular dichroism (Table 1) revealed that E. coli Hfq contains 30% ␤ strand content (31 residues), which nearly accounts for the number of residues necessary to form the canonical five-stranded Sm structure. Furthermore, the 10% ␣-helical structure of Hfq (10 residues) is in accord with the 10–11 residue ␣ helix found in the SmAp protein (Mura et al., 2001). Hfq Cooperates in RNA-RNA Interaction It seems plausible, based on the results described above, that Hfq could cooperate in formation of gene regulatory RNA-RNA complexes. To test this idea, we investigated complex formation of Spot 42 RNA to its target region in the gal mRNA (i.e., to the galK translational initiation region) using a gel mobility assay. The results are illustrated in Figure 6A. When a constant amount of 5⬘ end-labeled Spot 42 RNA (0.5 pmol) was incubated with increasing amounts of an unlabeled 310 nucleotide galK⬘ RNA substrate, which carried the galK Shine-Dalgarno sequence, 50% complex formation was

Molecular Cell 26

Figure 4. The Sm1 Sequence Motif Is Conserved in Hfq Proteins Amino acid sequence alignment of a subset of bacterial Hfq proteins. The Sm consensus based on 80 Sm and Sm-related proteins (Achsel et al., 1999) is shown at the bottom together with the amino acid sequence and secondary structure of the Archaeoglobus fulgidus Sm1 protein (residues forming the uracil binding pocket are labeled “#”) (To¨ro¨ et al., 2001). The Sm1 sequence motif of Hfq proteins is highlighted in gray. Positions that are identical in most Sm motifs are shown in bold in the Sm consensus. “h” signifies a bulky hydrophobic residue (L, I, M, V, F, Y, or W), and “ⵑ” signifies segment of variable sequence and length.

observed at an RNA concentration of ⵑ500 nM (lane 4). Addition of Hfq to the incubations strongly enhanced complex formation between Spot 42 RNA and galK⬘ RNA (lanes 5–8). Thus, at a galK⬘ RNA concentration of 20 nM more than 95% of the Spot 42 RNA-Hfq complex was shifted into a Spot 42-Hfq-galK⬘ complex (lane 6). Additionally, mobility assays revealed that Hfq at a monomer concentration of 1 ␮M improved complex forma-

tion more than 150-fold. Furthermore, Hfq formed complexes with the galK⬘ RNA, however its affinity for the galK⬘ RNA was considerably lower than that for Spot 42 RNA (data not shown). In order to learn more about the mechanism of Spot 42 RNA action, we carried out structural probing with the single-strand-specific endonuclease RNase T2. Previous probing of the Spot 42 RNA-galK⬘ RNA complex with this enzyme indicated that the bimolecular RNARNA interaction is mediated by tripartite interactions between exposed sequences of Spot 42 RNA and separated target sequences (Figure 7) (T. Møller et al., submitted). In keeping with this result, the structural probing revealed that the addition of galK⬘ RNA to incubations containing a fixed amount of 5⬘ end-labeled Spot 42 RNA reduced cleavage by RNase T2 in the 5⬘ tail, the top loops of the first hairpin, and at positions 47–48 and positions 57–60 of Spot 42 (Figure 6B, lanes 1–5). When Hfq was present, a very similar cleavage pattern was observed with the exception of the region of nucleotides 57–64 which became inaccessible or only weakly accessibly to T2 cleavage (lanes 6–10) (i.e., the region that bears an Sm-like site). However, protection of Spot 42 RNA was observed at much lower concentrations of the galK⬘ RNA (ⵑ100-fold). Based on these results, we infer

Table 1. Circular Dichroism Analysis of E. coli Hfq

Figure 5. Hfq Forms a Doughnut-Shaped Structure Rotary shadowed electron micrograph of purified E. coli Hfq. Note, rotary-shadowed particles are covered with carbon and platinum, and therefore such particles appear larger than negatively stained particles.

Structure

H

A

P

T

O

Total

% SD %

10 1

26 1

4 1

22 1

37 0

100

The circular dichroism spectra of Hfq in 200 mM NaF, 20 mM NaH2PO4 (pH 7.4) was recorded between 260 and 180 nm at 20⬚C. Secondary structures were determined as described by Compton et al. (1987). Helixes (H), antiparallel ␤ sheet (A), parallel ␤ sheet (P), turns (T), aperiodic structures (O), and standard deviation (SD).

Hfq: An Sm-Related Protein 27

Figure 7. Protein-Mediated RNA-RNA Interaction (A) Secondary structure of Spot 42 RNA. The galK complementary regions involved in base pairing are shown in a gray background. (B) Sequence of the translational initiation region of galK. The Spot 42 RNA complementary regions involved in duplex formation (Figure 6B) (T. Møller et al., submitted) are shown in a gray background, and Sm-like sites are indicated in bold. The galT stop codon and the galK start codon are underlined.

that Hfq strongly cooperates in RNA-RNA interaction between Spot 42 RNA and its target RNA, and moreover, Hfq does not appear to interfere with the structure of Spot 42 RNA.

Discussion Figure 6. Hfq Cooperates in RNA-RNA Interaction (A) Incubations containing 5⬘ end-labeled Spot 42 RNA (5 nM), 2 ␮M tRNA, and increasing amounts of unlabeled galK⬘ substrate were incubated in the absence (lanes 2–4) or in the presence of Hfq (lanes 6–8), and complex formation was monitored in a electrophoretic mobility shift experiment. galK⬘ RNA concentrations were 20 nM (lanes 2 and 6), 100 nM (lanes 3 and 7), and 500 nM (lanes 4 and 8), and the Hfq “monomer” concentration was 1 ␮M. Unbound Spot 42 RNA (I) and complexes corresponding to Hfq-Spot 42 RNA (II), Spot 42 RNA-galK⬘ RNA (III), and Spot 42 RNA-Hfq-galK⬘ RNA (IV) are indicated. (B) Nuclease probing of Spot 42-RNA-galK⬘-RNA and Spot 42-RNAHfq-galK⬘-RNA complexes. Incubations containing 5⬘ end-labeled Spot 42 RNA (5 nM), 3 ␮M tRNA, and increasing amounts of unlabeled galK⬘ substrate were incubated in the absence (lanes 1–5) or in the presence of Hfq (lanes 6–10) and treated with RNase T2. Lanes marked T1, OH⫺, and P are RNase T1 cleavage of Spot 42 RNA under denaturing condition (G-specific cleavage), alkaline hydrolysis ladder of Spot 42 RNA, and untreated Spot 42 RNA probe, respectively. The galK⬘ RNA concentrations for lanes 1–5 were 0, 100, 500, 1000, and 2000 nM, respectively, and for lanes 6–10 were 0, 4, 20, 100, and 500 nM, respectively. The Hfq “monomer” concentration was 1 ␮M (lanes 6–10). Arrows indicate reduced cleavage. Black bar indicates nucleotides that are inaccessible or only weakly accessible to T2 cleavage in the presence of Hfq.

Previous work established that the E. coli Hfq protein interacts with different RNAs and affects various aspects of RNA metabolism and thus shares functional features with members of the large family of eukaryotic and archaeal Sm and LSm proteins. Here we demonstrate that Hfq is structurally related to the Sm proteins and that a large number of bacteria encode this protein. As the archaeal AF-Sm1 and AF-Sm2 proteins, Hfq forms homomeric ring-shaped structures, and as the eukaryotic LSm proteins, Hfq oligomerizes into such structures in the absence of target RNA (Achsel et al., 1999, 2001). The Hfq proteins, however, are dissimilar to the eukaryotic and archaeal relatives in that they lack an obvious Sm2 sequence motif and are likely to form hexamers rather than heptamers. Despite these structural differences, E. coli Hfq appears to possess RNA binding specificity very similar to that of the Sm proteins. Moreover, Hfq strongly increases the affinity of the riboregulator Spot 42 RNA to its target in the gal mRNA. Below we discuss these findings in the context of antisense regulation as well as the functional implications for RNA-RNA complex formation in general.

Molecular Cell 28

RNA Determinants for Hfq Binding The Sm proteins bind a single-stranded U-rich sequence known as the Sm site (consensus Pu AU4–6 G Pu), which is often found between stem-loop structures in the spliceosomal UsnRNAs (Branlant et al., 1982). The sequence specificity is provided directly by the oligo(U) tract of the Sm site, and a synthetic Sm site or a short oligo(U)-nucleotide suffices for the formation of heptameric complexes (Raker et al., 1999; Urlaub et al., 2001). Similarly, the LSm2–LSm8 proteins interact with the oligo(U) tail of U6snRNAs (Achsel et al., 1999). Our probing of Spo42 RNA by hydroxyl radicals established that Hfq binding caused protection of three small U-rich segments in single-stranded regions of Spot 42 RNA (Figure 3A), and it appears likely that a single hexameric Hfq molecule is responsible for the protection of the three regions. Thus, in gel mobility shift assays Spot 42 RNA and Hfq just form one retarded complex (Figure 2A). Most strikingly, the protected regions of Spot 42 RNA all have sequences that resemble the Sm site (i.e., GAU3GG, AAUAU4AG and GU6A), suggesting that Hfq also possesses a RNA binding site with specificity for oligo(U) stretches. Inspections of the secondary structure of two other antisense RNAs that interact with Hfq further strengthen this view. Thus, DsrA RNA and OxyS RNA contain in single-stranded regions flanked by stem-loop structures either a near canonical Sm site (AAU6AA) or two related sequences (AGU3CUCAA and AACU4G) (Zhang et al., 1998; Lease and Belfort, 2000). We note that only a few of the residues that form the uracil binding pocket of the (AF-Sm1)-RNA complex (marked “#” in Figure 4) are conserved in the Hfq proteins (To¨ro¨ et al., 2001). Therefore, either a different mode of binding is used by the bacterial Sm-like proteins or residues K56H57-A58 function analogously to AF-Sm1 residues R63G64-D65 in RNA binding (Figure 4). Protein-Mediated RNA-RNA Interaction The three well-characterized E. coli sRNAs (i.e., OxyS, DsrA, and Spot 42 RNA) that act through base pairing are all trans encoded and, consequently, antisense-target complementarity is incomplete, and regulation must be achieved by formation of small, imperfect duplexes (Altuvia and Wagner, 2000). It is therefore difficult to understand how efficient binding and regulation can be established. This “paradox” is likely to be resolved by the finding that Hfq greatly enhances the affinity of Spot 42 RNA for its target in the gal mRNA. As recent studies indicate that Hfq also enhances OxyS RNA binding to the fhlA and rpoS mRNAs (Zhang et al., 2002 [this issue of Molecular Cell]) and DsrA RNA and RprA RNA regulation of rpoS mRNA requires Hfq (Majdalani et al., 2001; Sledjeski et al., 2001), we infer that Hfq acts as a general cofactor for antisense RNAs that rely on short base pairing by cooperating in complex formation. Moreover, we believe that our results are of general significance and might possibly aid the understanding of the role of Sm and LSm proteins in the splicing reaction. In this context, we note that recent studies in yeast indicate that three of the Sm proteins of U1snRNP make direct contact with the pre-mRNA substrate in the commitment complex, thereby contributing to complex formation and possibly also to the splicing reaction (Zhang et al., 2001).

Although the mechanism of how Hfq cooperates in complex formation warrants further investigation, any model must be able to account for the following observations: (i) Hfq interacts with Spot 42 RNA and the galK⬘ substrate, and when all three molecules are present, they form a nucleoprotein complex (Figure 6); (ii) formation of complexes between Hfq and Spot 42 RNA, as well as between Spot 42 RNA and galK⬘ RNA, are likely to involve tripartite interactions (Figures 3, 6B, and 7); (iii) the three target regions in the galK⬘ RNA substrate are all flanked by Sm-like sites (Figure 7); (iv) Spot 42 RNA sequences (i.e., nucleotides 55–61) involved in base pairing are also bound by Hfq (Figures 3, 6B, and 7); and (v) Hfq cooperates in complex formation and appears to facilitate RNA-RNA interaction (Figure 6). On this basis, we propose that Hfq and Spot 42 RNA bind synergistically to the target RNA, forming a nucleo-protein complex that is held together by multiple RNA-RNA and RNA-protein interactions. In addition, in order to supplement suboptimal RNA-RNA interactions with protein-RNA interactions, Hfq could possible directly facilitate RNA-RNA interaction by aligning complementary sequences on its surface. Electron microscopy of Hfq binding to bacteriophage Q␤ plus strand RNA supports such a binding feature (Miranda et al., 1997). Thus, Hfq seems capable of forming looping complexes, indicating that the protein can interact simultaneously with multiple targets and bring distal binding sites into close proximity of each other. Concluding Remarks This work gives credence to the assumption that the Sm protein family evolved from a common ancestor (Salgado-Garrido et al., 1999; Achsel et al., 2001), and it seems probable that other members of this family function in a manner similar to that of Hfq. We have speculated that the development of an ancestral heatstable Sm protein with specificity for U-rich sequences played a decisive role in evolution by modulating the stability, folding, binding, and indeed functions of RNA. Clearly such a protein would provide the cell with a simple device for establishing flexible bimolecular RNA interaction via short stretches of base pairing and thereby for the “construction” of simple, versatile RNAbased gene regulatory systems. As illustrated by present day riboregulators, any, or nearly any, short segment of a small RNA that is exposed can be exploited for target recognition (Altuvia and Wagner, 2000). Moreover, it is likely that the presence of specialized Sm proteins that carried extra subdomains were a prerequisite for the establishment of a primitive eukaryotic cell. Further studies of Hfq are important for several reasons. A full understanding of its structure and mechanism of action would provide general insights into numerous transactions on mRNA. Given the presence of Sm-like proteins in all organisms, protein-mediated antisense regulation could be extremely useful for medical and biotechnological purposes. Experimental Procedures Bacterial Strains All strains used were E. coli K-12 derivatives: SØ928 (⌬deo, ⌬lac) (Valentin-Hansen et al., 1978); NU426 (hfq⫹), TX2822 (hfq1), and TX2761 (hfq2) are described in Tsui et al. (1994).

Hfq: An Sm-Related Protein 29

Half-Life Determination Cultures were grown in Luria-Bertani medium at 30⬚C to an OD450 of 0.4 and treated with rifampicin (300 ␮g/ml). At the indicated times, 30 ml samples were collected by centrifugation at 0⬚C. Total RNA purification and Northern blot analysis was done as described (Franch et al., 1997, 1999) Purification of Hfq The intein system (Impact-CN TM, New England Biolabs, Beverly, MA) was used for Hfq purification. The E. coli hfq was amplified by PCR using oligonucleotides EC-N (5⬘-GGTGGTTGCTCTTCCAA CATGGCTAAGGGGCAATCTTTACAAGATC-3⬘) and EC-C (5⬘-TTAT TCGGTTTCTTCGCTGTCCTGTTG-3⬘) as primers and SØ928 chromosomal DNA as template. S. aureus hfq was amplified using oligonucleotides SA-N (5⬘-GGTGGTTGCTCTTCCACCATGATTGCAAAC GAAAACATCCAAG-3⬘) and SA-C (5⬘-CAACTTATTCTTCACTTTCAG TAG-3⬘) as primers and chromosomal DNA as template. PCR products were digested with SapI and cloned into SapI-SmaI digested pTyb11. Protein purification was done according to the manufacturers recommendations from strain ER2566. The lysis/wash buffer used was 20 mM Na-HEPES [pH 8] containing 500 mM NaCl. Gel Shift Analysis Spot 42 RNA and galK⬘ RNA synthesis and 5⬘ end labeling was done as described (T. Møller et al., submitted). 5⬘ end-labeled Spot 42 RNA, tRNA (Boehringer Mannheim, Mannheim, Germany), Hfq, and unlabeled Spot 42 RNA were mixed (as indicated) in 10 ␮l binding buffer (20 mM Na-HEPES [pH 8], 100 mM KCl, 1 mM DTT, and 1 mM MgCl2) and incubated on ice for 20 min. 5 ␮l 10% glycerol was added to the binding reactions immediately before loading onto a 5% nondenaturing polyacrylamide gel. After electrophoresis at 4⬚C, the gel was dried and subjected to autoradiography.

Electron Microscopy Purified E. coli Hfq was subjected to rotary shadowing electron microscopy as described (Morris et al., 1986). Briefly purified Hfq was sprayed onto freshly cleaved mica. The mica was transferred to an (Balzers BAE 250) evaporator and coated with a mixture of platinum and carbon. The surface replica was floated free from mica and transferred to grids in preparation for examination. Purified S. aureus and E. coli Hfq were used to prepare negatively stained electron microscope grids in 2% uranyl formate. Images were taken with a Philips 410 transmission electron microscope. Cross-Linking of Hfq 50 ␮l of E. coli Hfq (54 ng/␮l in 20 mM HEPES [pH 8], 200 mM NaCl, and 1 mM DTT) was made 0.1 M in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce, Rockford, IL) and allowed to react for 4 hr at room temperature. 5 ␮l of the reaction mixture was purified on a Poros R1 (Applied Biosystems, Framingham, MA) microcolumn. Elution was performed with 70% acetonitrile/water saturated with sinapinic acid (Fluka, Buchs, Switzerland) directly onto a MALDI target covered with a thin layer of sinapinic acid (Kussmann and Roepstorff, 2000). MALDI mass spectrometry was performed on a Voyager STR (Applied Biosystems, Framingham, MA) equipped with delayed extraction. Acknowledgments We would like to thank Udo Bla¨si for providing Hfq antisera and Hfq protein for the initial studies of Hfq-Spot 42 RNA interaction and Gisela Storz for sharing results prior to publication. This work was supported by grants from the Danish Natural Science Research Council (P.V.-H.), the Carlsberg Foundation (T.F.), and National Institutes of Health GM 492444 (R.G.B). Received September 24, 2001; revised November 19, 2001.

RNase T2 Probing of Spot 42 RNA Nuclease T2 digestion, alkaline hydrolysis, and RNase T1 cleavage of Spot 42 RNA were carried out as described in Franch et al. (1997, 1999). Immunoprecipitation 40 ml cultures were harvested at mid-log phase, and the cells were resuspended in 2 ml lysis buffer (200 mM K-glutamate, 20 mM Trisacetate [pH 7.25], 10 mM Mg-acetate, and 1mM DTT) and sonicated. After centrifugation, 0.5 ml of the extract was mixed with 5 ␮l Hfq antiserum, and the sample was rotated for 60 min at 4⬚C (fraction III). After 1 hr, 5 ␮l protein A agarose was added, and the sample was incubated for another 60 min. In a similar manner, 0.5 ml extract was mixed with 5 ␮l protein A agarose (control, fraction II) and incubated for 60 min at 4⬚C. Subsequently, the protein A agarose was collected by centrifugation and washed with lysis buffer. RNA isolated from protein A agarose pellets by phenol-chloroform extractions and ethanol precipitation was analyzed by Northern blot analysis. Also, total RNA was isolated from 0.5 ml of an untreated cell extract (fraction I). Hydroxyl Radical Footprinting 5⬘ end-labeled Spot 42 RNA (0.05 pmol) and the indicated amounts of purified Hfq were mixed in 10 ␮l binding buffer (20 mM Na-HEPES [pH 8], 100 mM KCl, 1 mM DTT, and 1 mM MgCl2) and incubated on ice. The samples were treated by a mixture of 0.5 ␮l 1.2% H2O2, 1.0 ␮l of EDTA/(NH4)2Fe(SO4)2 (4 and 2 mM, respectively), and 0.5 ␮l 20 mM ascorbic acid for 0.5 min. The reaction was terminated by addition of 20 ␮l 25% glycerol containing 0.5 ␮g/␮l of tRNA. After phenol extractions, the RNA was precipitated and analyzed on a 15% polyacrylamide sequencing gel. Circular Dichroism Circular dichroism spectra of E. coli Hfq in 200 mM NaF, 20 mM NaH2PO4 [pH 7.4] was recorded between 260 and 180 nm using a Aviv 202 spectropolarimeter. Secondary structure was determined using a variable selection method (Compton and Johnson, 1986).

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