1
Supplemental Methods
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Nickel Affinity Purification of His-tagged Hfqs
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E. coli cell pellets were lysed by lysozyme (200 µg/ml) and sonication (Lysis Buffer: 50 mM
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Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Triton X-100 + cOmplete Protease Inhibitor). Cell debris
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was removed by centrifugation and the supernatant was applied to a gravity flow column packed
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with nickel resin (Ni-NTA, Qiagen; ≈ 2 ml packed resin/1 L expression culture). The column
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was washed with 10-20 column volumes of each of the following: Equilibration Buffer (EB: 50
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mM NaH2PO4, 5 mM Tris-HCl, 300 mM NaCl, pH 8.0), EB + 1.5 M NaCl; EB + 40 mM
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Imidazole. Proteins were eluted in EB + 400 mM imidazole and stored in aliquots at -80 °C.
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For Hfq1, the 6xHis tag was removed by cleavage with His-tagged TEV protease (expressed
12
from pRK792, Addgene; Cambridge, MA). Imidazole was removed from His-Hfq1 by
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diafiltration into TEV Cleavage Buffer (50 mM Tris-HCl pH 8, 50 mM NaCl), and recovered
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protein was digested with a 1:5 molar ratio of TEV protease:His-Hfq1 for 2 h at 30 °C followed
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by 4 °C incubation overnight. A high protease to substrate ratio was utilized due to poor
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substrate cleavage, presumably due to aggregates. Uncleaved Hfq and His-TEV protease were
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removed by passage over nickel resin. For His-TEV-Hfq2, after dilution to < 40 mM imidazole,
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purified protein was reapplied to nickel resin, followed by washes with ≈ 20 column volumes
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Urea Buffer (50 mM Tris-HCl, 1 M NaCl, 2 M urea, 5% glycerol, pH 8.0) and ≈ 20 column
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volumes Glycerol Buffer (50 mM Tris-HCl pH 8.0, 1 M NaCl, 5% glycerol), followed by elution
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in Glycerol Buffer + 250 mM imidazole. For His-Hfq2, imidazole was removed by buffer
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exchange into Glycerol Buffer on a Zeba Spin gel filtration column (7 kDa MWCO;
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ThermoFisher, Waltham, MA).
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Additional AUC Protocol
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Samples of His-Hfq1 were studied at nominal loading concentrations of 120, 60 and 30 µM in
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500 mM NaCl and 20 mM Tris-HCl (pH 7.4) with an absorbance wavelength of 280 nm; Hfq1
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was studied at a nominal loading concentration of 8.3 µM in 500 mM NaCl and 50 mM Tris-HCl
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(pH 7.4) using an absorbance wavelength of 230 nm; His-Hfq2 in 500 mM NaCl and 20 mM
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Tris-HCl (pH 7.4) was studied at a single loading concentration at 260 nm; and His-TEV-Hfq2
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in 1.0 M NaCl, 50 mM Tris-HCl (pH 7.4) and 5% (v/v) glycerol was studied at 125 µM and 280
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nm. In all cases, data were analyzed in terms of a continuous c(s) distribution of sedimenting
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species in SEDFIT 14.4f1 using a resolution of 0.05S and a confidence interval of 0.68. As a
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result of the high solvent density, viscosity and refractive index, the analysis of the interference
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sedimentation data for His-TEV-Hfq2 required correction for a slight meniscus mismatch.2
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Protein partial specific volumes were determined based on their amino acid composition in
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SEDNTERP (http://sednterp.unh.edu/).3 Buffer densities and viscosities were determined in
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SEDNTERP based on their composition, except for 1.0 M NaCl, 50 mM Tris-HCl (pH 7.4) and
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5% (v/v) glycerol; for this buffer, these properties were measured experimentally at 20.00 °C on
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an Anton Paar DMA 5000 density meter and Anton Paar AMVn rolling ball viscometer.
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LC-MS Analysis
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For LC-MS analysis of Hfq1 and Hfq2, a small amount of His-tagged, purified protein (20 pmol)
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was injected on a 6130 Quadrupole LC/MS (Agilent Technologies, Santa Clara, CA) using
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acetonitrile-acetic acid as a mobile phase and a reversed-phase Poroshell column (Agilent
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Poroshell 300 SB-C3). The peak eluted at 60% acetonitrile, and mass spectra were analyzed
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using HP Chem Station deconvolution software to obtain intact molecular masses.
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Circular Dichroism Spectroscopy
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His-TEV-Hfq2 protein samples were analyzed directly in the high-salt Glycerol Buffer (50 mM
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Tris-HCl, pH 8, 1 M NaCl, 5% glycerol) in a 0.5 mm pathlength cuvette in a Jasco 700-series
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spectropolarimeter (Easton, MD). The spectrum was obtained from the average of 10 reads
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across the wavelength range of 200-260 nm.
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Fluorescence Polarization Assay
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Increasing concentrations of urea-stripped His-TEV-Hfq2 (to remove contaminating nucleic
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acids) in Glycerol Buffer + imidazole were distributed into wells of a black 96-well plate, using
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Glycerol Buffer + imidazole to bring the volume of all samples to 25 µl. Protein samples were
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mixed with 90 µl of 20 mM Tris-HCl (pH 8) containing 200 µg/ml BSA and 20 nM of either a 5’
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6-FAM-labeled A18 oligoribonucleotide or a 5’ 6-FAM-labeled oligoribonucleotide with the
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following sequence: 5’ AGAGAGAGAAGAGAGAGA (denoted as (AG)18). After incubation
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at room temperature for ≈ 30 min., fluorescence polarization was measured on a Victor 1420-040
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multilabel counter (λex = 485 nm, λem = 535 nm), and S and P values from a series of samples
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with protein only (no oligoribonucleotide) were subtracted before calculating the polarization in
64
units of milli-polarization (mP). For this preliminary assay, values are displayed without G-
65
factor correction.
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Additional Protein Modeling Protocol
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The templates for Hfq modeling were selected based on alignment length, highest sequence
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identity, and low gap content. Obtained models were refined by adjusting side chain rotamers
3
70
for agreement with the template structure. Hydrogens and charges were added to the models
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using the Amber ff12SB force field in Chimera 1.9,4 and they were energy minimized using 100
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steps of steepest descent and 30 steps of conjugate gradient minimization using Chimera’s
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standard parameter. Six models of each protein were assembled into a hexamer based on the
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template structures using the MatchMaker function of Chimera.
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Hexamer Mixing Experiments
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For Hfq1 + Hfq2 mixing experiments, lysates of IPTG-induced CEV001 and CEV002
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(prepared in lysis buffer by sonication; 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Triton X-
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100, 200 µg/ml lysozyme) were mixed in equal volumes at room temperature or at 4 ºC for 2
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hours, in the presence of 0-4 M urea. His-tagged proteins were recovered from the incubated
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mixture on a nickel column, using the general procedure described in Supplemental Methods,
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and analyzed by SDS-PAGE. Hfq2 + Hfq3 experiments were performed by simple mixing and
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incubation of purified His-TEV-Hfq2 and His-Hfq3 in 300 mM NaCl buffer with 0-2 M urea,
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followed by dilution of urea-containing samples to a final urea concentration of 1 M and direct
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separation of the mixed samples by SDS-PAGE.
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Supplementary Figure 1. Alignment of Hfq Protein Sequences in the B. cereus sensu lato
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Group. (A)-(B) Hfq1 and Hfq2 protein sequences from B. anthracis (B.a.), B. cereus (B.c.),
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and B. thuringiensis (B.t.) were aligned by ClustalW, and alignments are displayed by Boxshade.
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Hfq2 protein sequences are completely conserved among the group, with only small variations in
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Hfq1 protein sequences among the group. (C) Alignment of B. anthracis Hfq protein sequences
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against S. aureus Hfq as a reference sequence, as described in Fig. 1C-D.
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Supplementary Figure 2. Alternative Phylogenetic Tree. Unrooted phylogenetic tree
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constructed from an alignment of the Hfq protein sequences in Fig. 2A, using the Phylogeny.fr
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tool,5 which encompassed the use of the Gblocks (Curation), PhyML (Phylogeny by Maximum
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Likelihood), and TreeDraw (Tree Rendering, using branch lengths) tools. B. anthracis Hfqs
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indicated with arrows.
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Supplementary Figure 3. Additional Analysis of His-Hfq1 and His-Hfq2 Proteins. (A)
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Left panel, Semi-native gel (16% Tricine) of His-Hfq1 (this particular gel depicts the I39F
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mutant). Lanes 1-4 depict decreasing SDS concentrations in the loading buffer: Lane 1, 4%,
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Lane 2, 2%, Lane 3, 0.8%, Lane 4, 0.4%. Tricine running buffer included a constant [SDS]
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(0.1%) in each case. Right panel, 16% semi-native Tricine gel depicting His-Hfq1 and His-Hfq2
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separated on the same gel. (B) Sedimentation velocity c(s) analysis of His-Hfq2, as discussed in
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Fig. 4C. Absorbance data collected at 260 nm are shown here to highlight the presence of
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nucleic acid-containing Hfq complexes. (C) Fluorescence polarization measurements of purine
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oligonucleotides (A18, circle; and (AG)18, square; as described in Suppl. Methods) mixed with
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increasing concentrations of His-TEV-Hfq2 protein. Data points reflect the average of two
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replicates. (D) CD spectrum of His-TEV-Hfq2. Raw ellipticity was converted into molar
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residue ellipticity using the protein concentration as determined by measurement of the protein
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concentration by absorbance at 280 nm and the calculated extinction coefficient. Due to the
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aggregative propensity of the sample (and the associated use of high salt buffer), the signal
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amplitude is lower than expected, presumably due to loss of signal by backscatter. However, a
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shape consistent with α-helical protein content, combined with some β-sheet content, is
5
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observed. (E) Sedimentation velocity c(s) analysis of His-TEV-Hfq2, as discussed in Fig. 4D.
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Absorbance data collected at 280 nm are shown to highlight the disperse nature of the
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preparation.
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Supplementary Figure 4. Additional Analysis of His-Hfq1 and His-Hfq2 Proteins.
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(A) Mass spectrum and components for His-Hfq1. The primary peak matches the expected size
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of the methionine-cleaved product, with a secondary species consistent with His-Hfq1 with an N-
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formylated methionine. (B) DLS analysis of His-Hfq1, as continued from Fig. 3B. Protein was
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incubated with each chemical, as indicated, overnight before measurement. (C) Mass spectrum
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and components for His-TEV-Hfq2. The primary peak matches the expected size of the
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methionine-cleaved product, with a secondary species consistent with His-TEV-Hfq2 with an α-
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gluconylation, a common mutation for highly expressed recombinant products in E. coli.6
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Supplementary Figure 5. Supplemental In vivo Complementation Experiments. (A) The
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parent Δlac rpoS-lacZ (SG30013) and ΔlacΔhfq rpoS-lacZ (YN585) strains, as described in
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Methods and Fig. 7, were streaked to MacConkey agar (w/o ampicillin) as a color reference. #1-
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3, SG30013 colonies; #4-6, MG1655 E. coli parent strain colonies; #7-9, YN585 colonies. (B)
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Protein gel of denaturing cell lysates from the strains depicted in Fig. 7A (left side; CEV017-
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CEV019). Lanes 1-2, Hfq1; Lanes 3-4, Hfq2; Lanes 5-6, Hfq3; Lane 7, control cells without
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plasmid. Replicate lanes represent cells removed from separately streaked patches. Note that the
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high apparent level of His-Hfq2 expression suggests that the low level of purified protein
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recovery from the T5 construct (Fig. 3D) could be due to inclusion body formation or, possibly,
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degradation. (C) Impact of Q8A on His-Hfq1 interference (strain CEV029). Vector control
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indicates mutagenized Hfq1 vector that had lost its Hfq insert. (D) Protein gel of denaturing cell
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lysates from strain CEV020 (SG30013 + His-Hfq1 wild-type; Lane 1) and CEV026 (SG30013 +
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His-Hfq1-V37K; Lanes 2, 3). (E) Protein gel of denaturing cell lysates from strains CEV022
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(SG30013 + His-Hfq3 wild-type, Lane 1); CEV019 (YN585 + His-Hfq3 wild-type, Lane 3);
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CEV027 (SG30013 + His-Hfq3-Q8A, Lane 2); and CEV028 (YN585 + His-Hfq3-Q8A, Lane 4).
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Lane 5 is the His-Hfq1-2 chimera, provided as a size reference. (F) Alignment of two Hfqs that
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can complement Hfq function in E. coli: C. difficile Hfq and B. anthracis Hfq3, as compared to
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the alignment with B. subtilis Hfq.
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Supplementary Fig. 6. Analysis of Hfq Mixing Experiments. (A). Overexpression lysate
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mixing experiment between His-Hfq1 (red arrow) and His-Hfq2 (blue arrows), with urea
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treatments as indicated; 4-20% Tris-glycine. (B). In vitro purified protein mixing experiment
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between His-TEV-Hfq2 (purple arrows) and His-Hfq3 (green arrow), with urea treatment as
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indicated; 16% Tricine. In (B), the sample of His-Hfq3 available exhibited lower levels of
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hexamer formation under these conditions. Samples on each gel were run as boiled and unboiled
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in SDS loading buffer as indicated. Arrows denote the location of His-Hfq1, His-Hfq2, His-
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TEV-Hfq2, and His-Hfq3 purified proteins, as determined on a reference gel with purified
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samples.
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