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Acta Crystallographica Section F: Structural Biology and Crystallization Communications logoLink to Acta Crystallographica Section F: Structural Biology and Crystallization Communications
. 2010 Apr 30;66(Pt 5):563–566. doi: 10.1107/S1744309110009942

Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer

Seiki Baba a, Tatsuhiko Someya b, Gota Kawai c,d, Kouji Nakamura b, Takashi Kumasaka a,*
PMCID: PMC2864693  PMID: 20445260

The RNA-binding protein Hfq from B. subtilis was crystallized using the hanging-drop vapour-diffusion method in two crystal forms that belonged to space groups I422 and F222; diffraction data were collected to 2.2 Å resolution from both forms.

Keywords: RNA chaperones, gene-expression regulation, RNA-binding proteins, Hfq

Abstract

The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq–RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq–RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å, while the type 2 Hfq–RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å. Diffraction data were collected to a resolution of 2.20 Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis.

1. Introduction

Hfq is an important component in the regulation of gene expression according to changes in the growth environment and is found in many bacteria. This protein was first identified as a host factor that was required for the replication of RNA phage Qβ in Escherichia coli (Franze et al., 1972 ▶). The function of Hfq as a post-transcriptional regulator has been well studied in Gram-negative bacteria, especially E. coli; its regulation is controlled by interaction with various RNAs. E. coli Hfq (EcHfq) has been shown to interact with several small untranslated RNAs such as OxyS, DsrA and Spot42 (Zhang et al., 2002 ▶; Majdalani et al., 1998 ▶; Møller et al., 2002 ▶). Moreover, EcHfq can interact with many targets as an RNA chaperone for mRNA (Moll et al., 2003 ▶). In addition, EcHfq is also involved in the stabilization of RNA by protecting it from degradation by two exoribonucleases: RNase II and polynucleotide phosphorylase (Afonyushkin et al., 2005 ▶; Mohanty et al., 2004 ▶). In terms of gene regulation, EcHfq has been shown to interact with two RNA sequences: poly(A) tails of mRNA and unstructured A/U-rich sequences near stem-loop structures (Hajnsdorf & Régnier, 2000 ▶; Folichon et al., 2005 ▶; Brescia et al., 2003 ▶; Geissmann & Touati, 2004 ▶; Brennan & Link, 2007 ▶). Its RNA-­binding sites have been identified by site-directed mutagenesis (Mikulecky et al., 2004 ▶; Ziolkowska et al., 2006 ▶).

Recently, crystallographic analyses of Hfq from several different bacteria have revealed that it forms a homohexameric ring consisting of an Sm-like motif and has two possible RNA-binding sites on both sides of the torus-shaped ring (Schumacher et al., 2002 ▶; Sauter et al., 2003 ▶; Nikulin et al., 2005 ▶; Nielsen et al., 2007 ▶). The first structural determination of an Hfq–RNA complex was performed on Hfq from the Gram-positive Staphylococcus aureus (SaHfq) in complex with the short U-rich RNA AU5G; it revealed that the RNA is recognized by residues on the loop between β2 and β3 and β4 and β5, defined as proximal RNA-binding sites (Schumacher et al., 2002 ▶). Alternatively, a structure with RNA bound to the distal site has recently been determined for EcHfq (Link et al., 2009 ▶). This study showed that the poly(A) RNA binds to the (rA-rR-rN) RNA-binding site of EcHfq, on which the phosphodiester backbone traces a circular weaving pattern. This site consists of an adenosine-specific site, a purine nucleotide-selective site and a sequence-nondiscriminating RNA-entrance/exit site.

In the Gram-positive bacterium Bacillus subtilis, Hfq is encoded by the ymaH gene. B. subtilis Hfq (BsHfq) can bind both SR1 sRNA and ahrC mRNA; however, it was not required for stabilization of SR1 sRNA or for promotion of the RNA–RNA interaction between SR1 sRNA and ahrC mRNA (Heidrich et al., 2006 ▶, 2007 ▶). Based on its sequence similarity to SaHfq, BsHfq is expected to have a hexameric structure. On the other hand, sRNA–mRNA interactions in S. aureus are decisive for regulation of gene expression; they do not require Hfq (Bohn et al., 2007 ▶). We identified that BsHfq recognizes the A/G-­repeat RNA sequence as a novel Hfq-binding sequence using systematic evolution of ligands by exponential enrichment (SELEX) and gel-shift assays (manuscript in preparation). In order to investigate the RNA-binding mechanism, structural analysis of the BsHfq–RNA complex is in progress. Here, we report the expression, crystallization and preliminary X-ray studies of the complex of BsHfq with A/G-repeat RNA.

2. Materials and methods

2.1. Protein expression and purification

The coding sequence of Hfq from B. subtilis was amplified by PCR to add a BamHI site at the 5′ end and an EcoRI site at the 3′ end. Two primers were used: a forward primer (5′-CGCACTGGATCCATG­AAACCGATTAATATTCAGGATC-3′) containing a BamHI site (shown in italics) and a reverse primer (5′-CGCGTAGAATTC­TTATTCGAGTTCAAGCTGGACG-3′) containing an EcoRI site (shown in italics). The amplified fragment was cloned into a BamHI–EcoRI-digested pGEX-6P-1 expression vector (GE Healthcare). GST-Hfq was expressed in E. coli BL21; expression was induced for 5 h in the presence of 1 mM IPTG. Cells were harvested by centrifugation at 5000g and 277 K for 15 min. The wet cells were dissolved in sonication buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, 1 mM DTT pH 7.4), lysed on ice by sonication and centrifuged at 5000g and 277 K for 15 min. The supernatant was loaded onto a GST-affinity column (Glutathione Sepharose 4 Fast Flow, GE Healthcare) and eluted with 50 mM Tris buffer pH 8.0 containing 10 mM reduced glutathione and 1 mM dithiothreitol (DTT). The elution pattern was monitored by 15% SDS–PAGE. GST-Hfq-containing fractions were collected and dialyzed against 50 mM Tris buffer pH 7.0 and the sample was loaded onto an anion-exchange column (Q Sepharose FF, GE Healthcare) and eluted with 50 mM Tris buffer pH 7.0 containing 500 mM NaCl and 1 mM DTT; the elution pattern was monitored by 15% SDS–PAGE. The GST-Hfq-containing fractions were collected and concentrated by ultrafiltration with an Amicon Ultra-15 (Millipore). After GST-tag cleavage with PreScission protease (GE Healthcare), which leaves a remnant sequence (GPLGS) from the tag at the N-­terminus, the protein solution was loaded onto a HiTrap SP HP cation-exchange column (GE Healthcare) and eluted with a linear gradient of 100–400 mM NaCl in 35 ml 50 mM Tris buffer pH 7.0. The Hfq-containing fraction was pooled and concentrated using an Amicon Ultra-15. The protein concentration was determined by measuring the absorbance at 280 nm. The concentration of the purified protein was 8.9 mg ml−1 in 10 mM Tris–HCl pH 7.5, 10 mM NaCl.

2.2. RNA synthesis, purification and preparation

The RNA sample (rArGrArGrArGrA) was chemically synthesized using a DNA/RNA synthesizer (Expedite 8909, PerSeptive). The sample was purified using 20% PAGE under denaturing conditions with 8 M urea and was concentrated after desalting by ethanol precipitation. The solvent was adjusted to 10 mM Tris–HCl pH 7.5, 10 mM NaCl by adding concentrated buffer.

2.3. Crystallization

Crystallization was performed using the hanging-drop vapour-diffusion method at 293 K. Initial screening was performed with the commercial sparse-matrix crystallization kits Crystal Screen 1, Crystal Screen 2 and Natrix (Hampton Research). A 1 µl volume of Hfq–RNA solution was mixed with an equal amount of reservoir solution and the droplet was allowed to equilibrate against 300 µl reservoir solution in a sealed VDXm plate (Hampton Research). In the initial trial, crystals appeared after two weeks. After further optimization of the conditions, we obtained two crystallization con­ditions for Hfq–RNA: type 1 and type 2. In the crystallization con­dition for type 1 Hfq–RNA (714 µM Hfq protein:119 µM RNA) the reservoir solution consisted of 0.015 M cobalt(II) chloride, 0.1 M MES pH 6.5 and 1.8 M ammonium sulfate and the droplets were allowed to equilibrate against 100 µl reservoir solution in a sealed VDX48 plate (Hampton Research) for three weeks. For type 2 Hfq–­RNA (735 µM Hfq protein:250 µM RNA) the reservoir solution consisted of 0.01 M cobalt(II) chloride, 0.2 M MES pH 6.5, 1.8 M ammonium sulfate and the droplets were allowed to equilibrate against 300 µl reservoir solution in a sealed VDXm plate (Hampton Research) for two weeks. The molar ratios of the Hfq protein to the RNA aptamer in the crystallization conditions for type 1 and type 2 Hfq–RNA were 6:1 and 6:2, respectively.

2.4. Crystallographic data collection, processing and analysis

All X-ray diffraction data were collected on beamline BL38B1 of SPring-8 using a Rigaku Jupiter210 CCD detector. The type 1 and type 2 Hfq–RNA crystals were cryoprotected by soaking them in mother liquor with 24%(v/v) and 35%(v/v) glycerol, respectively. The crystals were then flash-cooled in a cold nitrogen-gas stream at 100 K. All data collections were performed at a wavelength of 1.0 Å with a total oscillation range of 180°. Diffraction images for type 1 and type 2 Hfq–RNA crystals were obtained with oscillation angles of 0.3° and 1.0°, exposure times of 40.0 and 20.0 s and crystal-to-detector distances of 150 and 176 mm, respectively. The data were integrated and scaled using the HKL-2000 program package (Otwinowski & Minor, 1997 ▶). MOLREP (Vagin & Teplyakov, 1997 ▶) was used to calculate self-rotation functions and perform molecular replacement using SaHfq (PDB code 1kq2; Schumacher et al., 2002 ▶), which shares 44% sequence identity with BsHfq, as a search model. The obtained models were refined with the program REFMAC5 (Murshudov et al., 1997 ▶). MOLREP and REFMAC5 were implemented within the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶).

3. Results

The type 1 crystals of Hfq–RNA (Fig. 1 ▶ a) were found to diffract to 2.20 Å resolution and to belong to the body-centred tetragonal space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å (Table 1 ▶). Assuming the presence of six protein monomers (one hexamer) in the asymmetric unit, the Matthews coefficient was 2.34 Å3 Da−1, corresponding to a solvent content of 48% (Matthews, 1968 ▶; Table 1 ▶). The type 2 crystals of Hfq–RNA (Fig. 1 ▶ b) were found to diffract to 2.20 Å resolution and to belong to the face-centred orthorhombic space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å (Table 1 ▶). Assuming the presence of three protein monomers (half a hexamer) in the asymmetric unit, the Matthews coefficient was 2.51 Å3 Da−1, corresponding to a solvent content of 51% (Matthews, 1968 ▶; Table 1 ▶).

Figure 1.

Figure 1

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(a) Type 1 and (b) type 2 Hfq–RNA complex crystals.

Table 1. Diffraction data statistics for Hfq–RNA complexes.

Values in parentheses are for the highest resolution shell.

  Type 1 Hfq–RNA Type 2 Hfq–RNA
X-ray source SPring-8 BL38B1 SPring-8 BL38B1
Detector Jupiter210 Jupiter210
Wavelength (Å) 1.0000 1.0000
Resolution (Å) 25.00–2.20 (2.28–2.20) 30.00–2.20 (2.28–2.20)
Space group I422 F222
Unit-cell parameters (Å) a = b = 123.70, c = 119.13 a = 91.92, b = 92.50, c = 114.92
Multiplicity 14.8 (14.9) 7.4 (7.4)
No. of observations 350287 92082
No. of unique reflections 23732 12626
Completeness (%) 100 (100) 100 (100)
Rmerge† (%) 6.3 (32.5) 3.3 (30.7)
Average I/σ(I) 38.02 (7.07) 45.75 (4.84)
Matthews coefficient (Å3 Da−1) 2.34 2.51
Solvent content (%) 48 51
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†

R merge = Inline graphic Inline graphic.

Self-rotation function calculations for type 1 and type 2 Hfq–RNA were performed using data in the resolution ranges 36.18–4.00 and 29.39–4.00 Å, respectively (Fig. 2 ▶). High peaks are observed for both crystal forms along the c axis at χ = 60° and 120° and correspond to sixfold and/or threefold noncrystallographic symmetry axes, respectively (Figs. 2 ▶ a and 2 ▶ c). By combination of the noncrystallographic and crystallographic symmetries, peaks for type 1 and type 2 Hfq–RNA around the c axis at χ = 180° (Figs. 2 ▶ b and 2 ▶ d) indicate that the sixfold noncrystallographic symmetry axis is parallel to the c axis in both forms. These results are consistent with the experimental assumption that BsHfq forms a homohexameric C 6 ring. This was confirmed by the molecular-replacement trial, as described below.

Figure 2.

Figure 2

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Self-rotation functions for (a, b) type 1 Hfq–RNA (I422) and (c, d) type 2 Hfq–RNA (F222). The sections corresponding to rotation by (a, c) χ = 60° and (b, d) χ = 180° are shown. This figure was produced using the program MOLREP (Vagin & Teplyakov, 1997 ▶).

Further molecular-replacement trials using the SaHfq structure gave good initial solutions with bound RNA. The solutions of type 1 and type 2 Hfq–RNA exhibited R factors of 0.335 and 0.328, and correlation coefficients of 0.631 and 0.650, respectively. Construction, revision and analysis of atomic models using the BsHfq sequence and RNA are currently in progress.

Acknowledgments

Synchrotron-radiation experiments were performed on beamline BL38B1 at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; proposal No. 2008B1506).

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