Purification of the large ribosomal subunit via its association with the small subunit

Analytical Biochemistry 395 (2009) 77–85

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Purification of the large ribosomal subunit via its association with the small subunit Samuel P. Simons a,1, Thomas J. McLellan a, Paul A. Aeed a,2, Richard P. Zaniewski b, Charlene R. Desbonnet b, Lillian M. Wondrack b, Eric S. Marr a, Timothy A. Subashi a, Thomas J. Dougherty b,3, Zuoyu Xu b,4, Ing-Kae Wang a,5, Peter K. LeMotte a,5, Bruce A. Maguire a,* a b

Department of Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA Department of Infectious Diseases, Pfizer Global Research and Development, Groton Laboratories, Groton, CT 06340, USA

a r t i c l e

i n f o

Article history: Received 22 June 2009 Available online 30 July 2009 Keywords: Ribosome 50S subunit Affinity purification Subunit association Complex purification Loose couples Tight couples

a b s t r a c t We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity. Ó 2009 Elsevier Inc. All rights reserved.

The complexity of the ribosome poses several challenges to purification. Traditional purification by ultracentrifugation is lengthy and laborious, and the three ribosomal RNAs (rRNAs)6 and more than 50 proteins that comprise the bacterial ribosome can easily be damaged or lost from the ribosome during the process. Chromatography can in theory overcome these limitations by providing rapid and gentle purification. However, although the established methods of gel filtration [1] and hydrophobic interaction

* Corresponding author. Fax: +1 860 441 6090. E-mail address: bruce.maguire@pfizer.com (B.A. Maguire). 1 Present address: Protein Sciences Corp., 1000 Research Parkway, Meriden, CT 06450, USA. 2 Present address: Pfizer Animal Health, Veterinary Medicine Research and Development, Kalamazoo, MI 49007, USA. 3 Present address: AstraZeneca Pharmaceuticals, Waltham MA 02451, USA. 4 Present address: Bacteriology and Mycology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 5 Present address: Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA. 6 Abbreviations used: rRNA, ribosomal RNA; HIC, hydrophobic interaction chromatography; EGTA, ethylene glycol bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; mRNA, messenger RNA; tRNA, transfer RNA; CAT, chloramphenicol acetyl transferase; RNase, ribonuclease; Ni–NTA, nickel–nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; CPM, counts per minute; LC–MS/MS, liquid chromatography–tandem mass spectrometry; HPLC, high-performance liquid chromatography; TFA, trifluoroacetic acid; ESI–TOF, electrospray ionization time-of-flight; SDS, sodium dodecyl sulfate; PDC, pyruvate dehydrogenase complex; SRP, signal recognition particle; GTPase, guanine nucleoside triphosphatase; CTC, catabolitecontrolled protein; EF, elongation factor. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.07.042

chromatography (HIC) [2] can purify and separate the large and small subunits, size exclusion is not easily scalable and requires prior sample concentration, whereas HIC requires salt concentrations that can damage ribosomes from less hardy bacterial species. Recently, a third method using cysteine–Sulfolink resin that lacks these drawbacks was described [3]. However, this method has proved to be unable to simultaneously separate the two subunits so that they must then be separated in a further step such as sucrose density gradient centrifugation. An alternative is to use one of the many tag-based purification systems now available. Tagged rRNA has been used to isolate ribosomes from cells, but because there are multiple copies of rRNA genes in most bacterial species, RNA tags are primarily useful to isolate a subpopulation of ribosomal subunits containing a tagged mutant rRNA [4–6]. Tagged ribosomal proteins have been used to purify mitochondrial ribosomes [7] for proteomic analysis. Tandem affinity tags have proved especially suitable to achieve the purity required for mass spectral analyses [8–10], but these employ large tags immunoglobulin G and require additives (EGTA) [11] that might affect ribosome structure and activity. Other tag-based purifications usually require changes in pH and ionic strength or the addition of competitor ligands such as imidazole and peptides for elution. These could potentially compromise ribosome structure or introduce unwanted contaminants. Furthermore, this form of purification often copurifies contaminating proteins that have a natural tendency to bind the resin, as routinely seen, for instance, with

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Purification of the large ribosomal subunit / S.P. Simons et al. / Anal. Biochem. 395 (2009) 77–85

His-tag purification. To avoid these complications, we devised an alternative elution strategy to purify 50S subunits from Deinococcus radiodurans. It was hoped that this would provide better quality 50S for crystallization to supply an X-ray crystallography program and do so more efficiently than existing centrifugation methods. Our method first captures the small (30S) subunit in order to purify the large (50S) subunit. Large subunits are captured via their association with the small subunit as 70S couples. Since this association is mediated largely by magnesium ions [12], reduction of the magnesium ion concentration dissociates the 50S subunits under mild conditions, yielding an extremely pure preparation free of the contaminants eluted by standard methods. There are two classes of 70S ribosomes distinguishable by the strength of their subunit association [13]: those that dissociate at a moderate magnesium concentration (‘‘loose” couples) and those that remain associated until a much lower magnesium concentration (‘‘tight” couples). These can be eluted separately by progressively reducing the magnesium concentration. We demonstrate efficient purification of highly pure 50S using a FLAG-tagged 30S protein to capture 70S ribosomes. The 50S subunits are unmodified, and only those functional enough to interact with the 30S can be selected. At the same time, the chromatography sorts the 50S into three classes according to their association status (unassociated, loosely associated, and tightly associated), improving homogeneity of the purified subunit for downstream applications. Rapid purification of yeast ribosomal subunits, ribosomes, and polysomes had already been demonstrated using a FLAG-tagged version of the yeast homolog of the bacterial ribosomal protein L23 [14]. This was used primarily to isolate associated messenger RNA (mRNA) but could be suitable for large-scale ribosome purification. A histidine tag was introduced simultaneously, but details of its use were not published and our own attempts to demonstrate the utility of this tag in the yeast strain were unsuccessful. We suspected that the six consecutive histidines of the tag might interact with rRNA, either electrostatically or by stacking, and so hinder binding to the nickel resin. However, another study in yeast was successful, perhaps because in that case the His tag was located at the end of a long protein stalk that protrudes from the large subunit’s surface. This stalk contains the P1/P2 protein complex, the components of which were individually tagged and used to dissect its stoichiometry [15]. However, possible use for large-scale subunit purification was not reported, and because the equivalent proteins in bacteria (proteins L7/L12) can easily be dissociated from the subunit [16], we speculated that this might make it an unreliable handle for capture. We chose the S16 protein of the 30S subunit as a suitable target for fusion with an affinity tag for several reasons. First, the X-ray crystallographic structure of the Thermus thermophilus 30S subunit (PDB file 1J5E) [17] shows that its C terminus is exposed on the lower part of the body of the subunit on the solvent side. This places it away from functional centers such as the binding sites for mRNA, transfer RNAs (tRNAs), translation factors, and the large ribosomal subunit. There is no crystal structure for the 30S subunit of D. radiodurans, but an alignment of its S16 sequence with that of T. thermophilus showed that its terminus aligns with the last amino acid of the T. thermophilus protein seen in the structure (Glu83). Second, the gene that encodes S16 in D. radiodurans is not part of an operon. This avoids possible effects on expression of other genes due to operon polarity, effects on mRNA structure or stability, or effects on the translational autoregulation that collectively regulates translation of ribosomal protein genes that share an operon [18]. Third, the Escherichia coli S16 protein has been shown to assemble into functional ribosomes even with a whole protein (RimM) appended to its C terminus [19], so the addition of a short tag should pose no problem.

There is precedence in the literature for the use of a FLAG tag, as noted above, for purification of yeast subunits. A version using a polyhistidine tag for purification was also generated because of the higher affinity and greater capacity of immobilized metal affinity chromatography resins. However, in view of our experience with the His-tagged yeast protein, we instead chose a naturally occurring variant of the His tag (HAT tag, Clontech, Palo Alto, CA, USA) that has the six histidines spaced out over a 19-amino-acid stretch.

Materials and methods Reagents Yeast extract was obtained from Difco, and tryptone was obtained from Oxoid. All other chemicals were purchased from J.T. Baker. Restriction enzymes were obtained from Roche Diagnostics or New England Biolabs. Gene replacement cassette A gene replacement cassette containing the D. radiodurans S16 coding sequence (rpsP, locus DR_1294, JCVI CMR database) preceded by upstream sequence that contains its promoter was synthesized as shown below (Blue Heron Biotechnology):

BamHI—upstream784bp—S16—NheI—FLAG=HAT-AscI— 432bp-CAT-FseI—downstream762bp—SalI: Sequence encoding a FLAG tag (DYKDDDDK) or a HAT tag (KDHLIHNVHKEEHAHAHNK) was added to the 3’ end of the S16 gene for expression as a fusion protein. The chloramphenicol acetyl transferase (CAT) coding sequence from plasmid pRAD1 [20], including its promoter, was included downstream to facilitate selection of positive clones by chloramphenicol resistance. Flanking sequences for homologous recombination (to replace the chromosomal S16 gene) were included on each end of the construct [21]. The cassette was cloned into pUCminusMCS (Blue Heron Biotechnology) for amplification in E. coli DH5a cells (Gibco), and DNA was linearized with BamHI and SalI for transformation into D. radiodurans. Transformation Deinococcus strain DSMZ 20539 was grown to confluence on blood agar and then scraped into TGY medium (10 g tryptone, 5 g glucose, 5 g yeast extract, and 5 g NaCl per liter at pH 7.2). Then 1.3 ml of cells at an A600 of 0.38 was diluted into 20 ml of TGY medium and grown to an A600 of 1. Cells were concentrated 10-fold, and CaCl2 was added to a final concentration of 30 mM [22,23]. Cells were incubated first at 30 °C for 80 min and then on ice with 1.2 lg of linearized DNA for 30 min. Transformed cells were diluted 1:100 for overnight growth, and 100 ll was plated on TGY plates containing 3 lg/ml chloramphenicol. Individual colonies from 6-day growth on TGY–chloramphenicol were streaked on blood agar plates and grown for 3 days. Cells were scraped from the plate using an inoculation loop, and one loopful was resuspended in 7 ml of TGY medium (A600 = 3.5). Then 4.2 ml of sterile 40% glycerol was added, and cells were frozen at 80 °C. Fermentation Cultures of FLAG–S16 or HAT–S16 D. radiodurans were grown in an IF-150 fermentor (New Brunswick Scientific) at 30 °C with

Purification of the large ribosomal subunit / S.P. Simons et al. / Anal. Biochem. 395 (2009) 77–85

400 rpm agitation and 20 L/min aeration. 2 L of actively growing overnight culture (in TGY medium containing 3 lg/ml chloramphenicol) was used to inoculate 38 L of growth medium (TGY supplemented with manganese sulfate monohydrate [0.82 mg/L] and no chloramphenicol) to A600 = 0.15. At A600 = 3, the culture was cooled to 15 °C over 15 min and approximately 180 g of cell paste was harvested by refrigerated continuous flow centrifugation (Heraeus Contifuge) and stored at 80 °C. Chromatography of FLAG-tagged ribosomes All procedures were done at 4 °C. Cells (20 g) were thawed in 40 ml of lysis buffer (10 mM Hepes–NaOH [pH 7.8], 30 mM MgCl2, and 150 mM NH4Cl), broken in a French press at 10,000 psi, and incubated on ice for 30 min with 2 ll per milliliter of deoxyribonuclease (ribonuclease-free; Roche Diagnostics). Unbroken cells were removed by centrifugation for 30 min at 18.5 Krpm in a Sorvall SS34 rotor, and the supernatant was filtered through a 0.22-lm filter (GP Express Plus Stericup, Millipore) to yield 37 ml containing 6500 A260 units. The sample was loaded onto a 100-ml column of anti-FLAG M2 resin (Sigma–Aldrich) at 0.5 ml/min on an ÄKTA basic chromatography platform (GE Healthcare). The column was washed with lysis buffer, and 50S subunits were eluted with a reverse magnesium ion gradient (A = lysis buffer; B = 10 mM Hepes– NaOH [pH 7.8], 0.25 mM MgCl2, and 100 mM NH4Cl). The 30S subunits were then eluted in the same buffer supplemented with FLAG peptide (100 lg/ml). Fractions containing 50S or 30S subunits were pooled, and their magnesium and b-mercaptoethanol concentrations were restored to approximately 10 and 6 mM, respectively, by dropwise addition with gentle agitation of 1/10 volume of elution buffer supplemented with 99 mM MgCl2 and 66 mM b-mercaptoethanol. Subunits were then pelleted for 14 h at 26 Krpm in a Beckman Type 45 rotor, resuspended in resuspension buffer (10 mM Hepes–NaOH [pH 7.8], 15 mM MgCl2, 75 mM NH4Cl, and 6 mM b-mercaptoethanol), flash frozen in liquid nitrogen, and stored at 80 °C. To isolate 50S subunits from the column flow-through, fractions comprising the second half of this peak were pooled and concentrated under nitrogen pressure in an Amicon stirred cell with a 62-mm YM100 membrane (Millipore) and then centrifuged on a 10 to 40% sucrose density gradient made in lysis buffer (with 6 mM b-mercaptoethanol) in a Beckman Ti-15 zonal rotor at 32 Krpm for 18 h. Fractions containing the 50S subunits were pooled and concentrated in a stirred cell, replacing the sucrose solution with buffer. The 50S were pelleted in a Beckman Type 65 rotor at 25 Krpm for 14 h and then resuspended in resuspension buffer and frozen. To polish purified subunits by pelleting through a sucrose cushion, 100 A260 units of purified 50S was diluted in 8 ml of lysis buffer supplemented with 0.5 M NH4Cl, underlaid with 2 ml of 1.1 M sucrose in the same buffer and centrifuged at 45 Krpm in a Type 65 rotor for 17 h. The pellet was resuspended in 1 ml of resuspension buffer, repelleted at 60 Krpm for 1.75 h in a Beckman TLA 100.2 rotor, resuspended in resuspension buffer, and clarified by microfuging for 15 min. Purification of 50S by centrifuge 50S subunits purified by centrifugation alone were used for comparative purposes in Figs. 4B and 5. These were purified by a novel sucrose density gradient procedure optimized for subsequent subunit crystallization that will be described in detail elsewhere (McLellan et al., submitted for publication). Use of a cysteine–Sulfolink precolumn for some of these preparations is described below.

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Cysteine–Sulfolink chromatography Lysates from wild-type (strain DSMZ 20539) or FLAG–S16 D. radiodurans were prepared as for FLAG chromatography, loaded onto a 125-ml column of cysteine–Sulfolink, and washed with 10 column volumes of lysis buffer. For subsequent FLAG chromatography of the tagged ribosomes, the column outflow was connected to the anti-FLAG column and then eluted with the same buffer but containing 0.3 M NH4Cl. For chromatography of wild-type cells, the lysis buffer included 6 mM b-mercaptoethanol and the column elution was concentrated in an Amicon stirred cell with buffer exchange into lysis buffer. The volume was reduced to 50 ml for subsequent purification by centrifugation according to the direct isolation protocol described by McLellan and coworkers (submitted for publication). Chromatography of HAT-tagged ribosomes Lysate from cells expressing HAT-tagged S16 was prepared as above. The sample was loaded onto a column of 65 ml of nickel– nitrilotriacetic acid (Ni–NTA) Superflow resin (Qiagen) equilibrated in lysis buffer as above. The column was washed with 10 column volumes, and 50S subunits were eluted from loosely and tightly associated 70S by lowering the magnesium ion concentration as described for the FLAG chromatography. The starting buffer conditions were restored, and 30S subunits were eluted with a gradient from 0 to 100 mM imidazole. Fractions containing the 30S subunits eluted between 10 and 40 mM imidazole and were pooled and concentrated 10-fold in a Millipore stirred cell (YM100 membrane) with buffer exchange to remove imidazole. The subunits were pelleted in a Type 45 rotor (19.5 h at 35 Krpm), resuspended in resuspension buffer, and flash frozen in liquid nitrogen. Various conditions were tested to improve binding of the 70S ribosomes; both Ni–NTA and Talon resins (BD Biosciences) were tested in lysis buffer, in resuspension buffer, or in the low ionic strength buffer G previously used to purify 70S ribosomal complexes containing tagged release factors on Ni–NTA resin [24]. Binding to Ni–NTA resin was tested further in lysis buffer supplemented (separately) with 10% glycerol, with b-mercaptoethanol at 4 or 6 mM, with imidazole at 5 or 10 mM (at pH 7.0 or 8.0), with KCl replacing the NH4Cl or the latter increased to 0.3 or 0.5 M. None of these conditions improved binding. Batch binding of HAT-tagged 30S and 70S Unbound ribosomes from the Ni–NTA column described above were loaded onto 10 to 40% sucrose density gradients made in lysis buffer for 70S isolation or in lysis buffer with 1 mM MgCl2 and 100 mM NH4Cl for subunit isolation. (Under the high hydrostatic pressure generated by ultracentrifugation, 1 mM magnesium is low enough to dissociate all of the 70S ribosomes into subunits.) The lysates were diluted twofold in the centrifugation buffer before loading, and the gradients were centrifuged at 4 °C in an SW28 rotor for 14.5 h at 23 Krpm and fractionated. Pooled fractions of 30S or 70S were concentrated in a stirred cell (YM100 membrane) to a final volume of 10 to 15 ml. The buffer was exchanged with lysis buffer to restore magnesium levels and remove sucrose before centrifuging at 35 Krpm for 3 h in a Type 65 rotor. Pellets were resuspended in lysis buffer (to 200 A260/ml) on a wrist action shaker. Then 6 A260 units of each sample was added to 200 ll of Ni–NTA Superflow resin in lysis buffer in a final volume of 0.6 ml of buffer. Samples (10 ll) of supernatant were withdrawn after brief microfuging to measure A260 immediately and then at intervals after incubation at 4 °C in an end-over-end tube rotator.

Purification of the large ribosomal subunit / S.P. Simons et al. / Anal. Biochem. 395 (2009) 77–85

Convex 10 to 50% sucrose density gradients were made in lysis buffer and centrifuged at 4 °C for 1.5 h in a Beckman SW55 rotor at 50 Krpm to allow visualization of polysomes. Linear 15 to 30% gradients were centrifuged at 50 Krpm for 2 h 20 min. Gradients were fractionated using an ISCO Type 11 optical cell and model UA6 absorbance monitor. Extraction and polyacrylamide gel electrophoresis (PAGE) of rRNA and proteins were performed as described previously [3]. polyU-directed in vitro translation 70S ribosomes were reconstituted by adding 1 A260 unit of 50S to a 2.4-fold excess of 30S (1.2 A260) in 10 ll of TMK buffer (10 mM Tris, 14 mM magnesium acetate, 60 mM KCl, and 1 mM dithiothreitol adjusted to pH 8.2 with acetic acid). The reaction buffer was 12 ll of translation buffer mix (Promega), S100 prepared from E. coli (110 lg protein by Bradford assay), 0.15 lCi phenylalanine 14 L-[ C(U)] (PerkinElmer NEC284E050UC), and TMK buffer to a volume of 30 ll. This was added to 10 ll of polyU (potassium salt Sigma P9528, dissolved in water at a concentration of 2.5 mg/ml) and 10 ll of ribosomes in a 96-well assay plate and incubated at 35 °C for 60 min. Then 5 ll of a bovine serum albumin solution (2 mg/ml in 10 mM Tris–HCl at pH 7.0) was added per well, followed by 36 ll of 0.833 N NaOH, and incubation continued at 35 °C for 15 min. Then 100 ll of stop solution (16% trichloroacetic acid [TCA] containing 2 mg/ml Difco casamino acids) was added to each well and the plate was incubated at 4 °C for 30 min. Reactions were then filtered using GF/B plates (prewet with 8% TCA) and a PerkinElmer FilterMate Harvester. Filtered assays were washed twice with 8% TCA and once with 95% EtOH. The filters were dried with a heat lamp, 60 ll of Packard MicroScint scintillation fluid was added per well, and counts per minute (CPM) was determined using a TopCount liquid scintillation counter (Packard). In-gel digestion of proteins and peptide identification by mass spectrometry Coomassie-stained protein bands were excised from the polyacrylamide gel, reduced, denatured, alkylated, and digested with trypsin (Promega), and peptides extracted as described previously [25]. The samples were analyzed using liquid chromatography– tandem mass spectrometry (LC–MS/MS) with subsequent database searching to identify the proteins contained in the gel bands. Peptides were separated by injecting 8 ll of sample via an Agilent capillary high-performance liquid chromatography (HPLC) system onto a Vydac C18 MS column (0.5  100 mm, 5 lm, 300 Å) at a flow rate of 5 ml/min. The mobile phases consisted of 0.2% trifluoroacetic acid (TFA) in water (A) and 0.2% TFA in acetonitrile (B). The peptides were separated using a linear gradient from 1.6 to 35% B over 98 min. The eluent was analyzed on a Thermo Fisher LTQ linear ion trap mass spectrometer outfitted with a PicoTip operating in positive ion mode using a data-dependent experiment for collection of MS/MS data with a dynamic exclusion of 3. Subsequent LC–MS/MS experimental data were converted into peak list (DTA) files and submitted to the Mascot protein database search engine. Searches were performed against the MSDB database while considering up to two missed cleavages with the mass tolerance for the monoisotopic peptide masses set to ±0.2 Da.

taining 1% b-mercaptoethanol was added, followed by 0.1 volume of 1 M MgCl2 and 2 volumes of acetic acid. Samples were incubated on ice for 45 min with occasional mixing and then microfuged for 15 min at 4 °C to pellet the RNA. The supernatant (containing 1 pmol 50S proteins/ll) was removed and stored at 20 °C. Prior to injection on the LC–MS, bovine RNase A was added (to 1 pmol/ll final concentration) as an internal standard for normalization to obtain relative quantitation between preparations. LC–MS was performed using an Agilent 1100 binary HPLC system complete with diode array with the effluent flowing into a Waters LCT electrospray ionization time-of-flight (ESI–TOF) mass spectrometer. The electrospray interface of the mass spectrometer was operated in the positive ion mode with a source block temperature of 100 °C and a desolvation temperature of 350 °C. The chromatographic separation was performed using a Vydac low TFA C4MS column (1.0  150 mm, 5 lm, 300 Å) maintained at 40 °C at a flow rate of 200 ll/min. Mobile phases were 0.05% TFA in water (A) and 0.0375% TFA in acetonitrile (B). The proteins were separated using a linear gradient from 10 to 50% B over 155 min.

Results Purification by FLAG affinity chromatography The chromosomal S16 gene in D. radiodurans was replaced with a version encoding a C-terminal FLAG or HAT tag. Cells were transformed with DNA encoding the tagged protein and the CAT gene to allow positive selection of recombinants (see Materials and methods). The gene replacement cassette was flanked with sequences that are upstream and downstream of the chromosomal S16 gene to promote its replacement by homologous recombination. Expression of the FLAG-tagged version of S16 in recombinants was confirmed by Western blotting with anti-FLAG antibody, and expression of the HAT-tagged version was confirmed by the appearance of a new peak with the correct mass (12,158.78 Da) in LC–MS whose identity was confirmed by tryptic digestion and LC–MS/MS. Cell lysates made from the FLAG–S16 D. radiodurans were subjected to anti-FLAG affinity chromatography. A typical chromatogram is shown in Fig. 1, with analysis of the ribosomal material in each elution peak by sucrose density gradient centrifugation shown in Fig. 2. Fig. 2A shows the filtered lysate that was loaded onto the column. This contains soluble proteins and tRNA at the top of the gradient, followed by free 30S and 50S subunits and then 6000

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Profiling of ribosomal proteins extracted from 50S subunits by LC–MS Proteins were extracted from 50S subunits as follows. First, 120 lg of 50S subunits (2.5 A260 units) was diluted to a volume of 6.25 ll in resuspension buffer. Then 20 ll of 10.5 M urea con-

Fig. 1. Chromatogram of a standard purification of FLAG-tagged D. radiodurans ribosomes. Absorbance is given by a solid line and Mg2+ concentration by a dotted line. Peaks of unbound material (flow-through) and 50S and 30S subunits are indicated. LC and TC denote 50S derived from loose and tight couple 70S, respectively. The introduction of the FLAG peptide is indicated by an arrow.

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Sedimentation Fig. 2. Absorbance traces from chromatographed samples centrifuged on sucrose density gradients. 30S and 50S subunits are labeled, as are 70S monosomes and disomes to pentasomes (2–5). (A) Column load. (B) Flow-through (containing free 50S). (C) 50S from loose couple 70S eluted at 1.75 mM Mg2+. (D) 50S from tight couple 70S eluted at 0.25 mM Mg2+. (E) 30S subunits eluted by FLAG peptide. A peak of 30S dimers is marked with an asterisk (). (F) flow-through containing free 50S from a similar column that was preceded by a cysteine–Sulfolink precolumn.

70S monosomes. Polysomes containing up to five 70S are resolved at the bottom of the gradient (labeled 2–5). Binding to the column results in capture of all but the free 50S and the low-molecular-weight contaminants seen at the top of the sucrose gradient (Fig. 2B). Separate analysis of different cuts from the flow-through peak suggested that a degree of size exclusion operates for the unbound material, with pigmented membranous material in early fractions followed by 50S subunits and then soluble proteins. After washing the column, the magnesium ion concentration was progressively lowered to dissociate first the loose couple 50S (at 1.75 mM magnesium) and then the tight couple 50S (at 0.25 mM magnesium). These concentrations are lower than those used for centrifugal separations (typically 6 and 1 mM magnesium, respectively) because the high hydrostatic pressures generated during ultracentrifugation assist dissociation [26]. The purity and identity of this material are confirmed by the sucrose density gradient profiles (Fig. 2C and D). There is a small peak of 70S detected in the loose and tight couple-derived 50S samples. However, because the 50S subunit contributes two-thirds of the absorbance (at 254 nm) of a 70S couple, this peak corresponds to only 1% contamination by 30S subunits. This low level of contamination suggests that each individual 70S monosome in a polysome binds independently to the resin so that few 30S subunits are released from them on dissociation by lowered magnesium or that those 30S subunits that do dissociate simply rebind to vacant antibodies on the resin.

The tagged 30S subunits were competed off the resin by buffer containing the FLAG peptide. The sucrose gradient (Fig. 2E) shows that this material sediments mainly at 30S, as expected, but it also contains an additional peak of 30S subunit dimers (marked with an asterisk) sedimenting between the 30S and 50S subunit positions. This marked tendency to dimerize was seen only for 30S purified by FLAG or HAT affinity chromatography and not for 30S purified by sucrose density gradient ultracentrifugation. The free 50S subunits in the flow-through fraction were purified away from the contaminating proteins and tRNA by preparative sucrose density gradient centrifugation (see Materials and methods). An alternative strategy was to use a cysteine–Sulfolink precolumn to remove these contaminants [3]. After washing, the cysteine–Sulfolink column was connected to the FLAG column and the ribosomes eluted directly into the second column with buffer containing 0.3 M NH4Cl. The free 50S subunits were collected in the flow-through from the FLAG column, and the loosely and tightly associated 50S and 30S eluted as before. The improved purity of the free subunits from such a tandem purification is demonstrated by sucrose density gradient centrifugation (Fig. 2F), but that of the other subunit elutions was not visibly changed. Final yields from 20 g of cells were 200 A260 units of free 50S, 540 units from loose couples, 630 units from tight couples, and 1100 units of 30S (one A260 unit corresponds to 69 pmol of 30S or 34.5 pmol of 50S). This yield accounts for the ribosomes in the original cell lysate. (Due to reduced cell breakage and perhaps a

Purification of the large ribosomal subunit / S.P. Simons et al. / Anal. Biochem. 395 (2009) 77–85

MW Total Free Loose _ " ++ _ " ++ Pre column: column: kb Pre 3,4 6 2 1.5 1

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the cell lysate so that no intact 23S remains. The same is true of rRNA from the loose and tight couples (lanes 5 and 7) unless a cysteine–Sulfolink precolumn was used (lanes 6 and 8). Therefore, 50S subunits are still prone to nuclease digestion during the FLAG chromatography, as they are during purification from this organism by sucrose density gradients [3], but the precolumn can help to prevent this. Smaller scale FLAG affinity purifications yielded rRNA that was more intact, presumably because separation of ribosomes from contaminating nucleases was more rapid. Both samples of 30S subunits (lanes 9 and 10) still contain some intact 16S rRNA, as does a sample of HAT-tagged 30S from a separate experiment (lane 11).

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Fig. 3. RNA extracted from purified subunits. The molecular weights of RNA markers in lanes 1 and 13 are shown, as are the positions of intact 16S and 23S rRNA in lanes 2 and 12. The source of the RNA in each lane is indicated above the gel.

Protein content Further differences are detected in the protein content of the various samples, shown by sodium dodecyl sulfate (SDS)–PAGE analysis in Fig. 4. The eight central lanes (lanes 3–10) contain relatively pure ribosomal proteins; all of these run below the 36-kDa molecular weight marker since only one ribosomal protein (protein S1) has a molecular weight above 36 kDa and this, dissociable protein, tends not to be recovered with 30S subunits from D. radiodurans. The spread of proteins in lane 2 shows, as expected, that the fraction of the cell lysate that fails to bind to the FLAG column comprises mainly nonribosomal proteins. Purification of the free 50S subunits from this fraction by centrifugation resulted in a sample (lane 3) containing mainly ribosomal proteins with a few contaminants such as a prominent band that migrates with the 97-kDa marker (labeled 1 on the figure). If a cysteine–Sulfolink precolumn is used to remove nonribosomal contaminants first, then the free subunits obtained from the FLAG column are much cleaner (lane 4). Samples of 50S subunits derived from loose couples either with or without the precolumn are the purest (lanes 5 and 6), whereas

lower content of ribosomes per cell, fewer ribosomes per gram of D. radiodurans cells are typically obtained than from E. coli.) Maximum binding capacity was approximately 1.4 mg (1.5 nmol) of 30S per milliliter of anti-FLAG resin. This is roughly fourfold less than expected for the average protein and likely reflects simple crowding or reduced accessibility to the antibody caused by the large size of the 30S (900 kDa). If the minimum amount of resin was used, the baseline of the chromatogram was higher, presumably due to leaching of bound ribosomes, so that an excess of resin was preferred to maintain subunit purity.

Integrity of rRNA rRNA was extracted from each sample of purified subunits and compared by PAGE. In lanes 2 and 12 of Fig. 3, the positions of intact 16S and 23S rRNA are marked. 23S rRNA extracted from free 50S subunits (lanes 3 and 4) has been cleaved by nucleases in

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Fig. 4. Proteins extracted from subunits purified on a FLAG column without or with a cysteine–Sulfolink precolumn. (A) SDS–PAGE. The first and last lanes are molecular weight markers. The second lane is column flow-through (FT) from a FLAG column alone, followed by 50S subunits purified by sucrose gradient centrifugation of this sample. The fourth lane is flow-through from a FLAG column with precolumn. Samples run in each lane are identified above the image by the type of subunit sample (free, loose, or tight 50S, or 30S) and whether a precolumn was employed. The last sample lane contains HAT-tagged 30S subunits purified by Ni–NTA chromatography. Contaminant bands identified by mass spectral analysis are marked as follows: (1) pyruvate dehydrogenase complex (PDC) E1 component, RNase (Accession No. AAF09933), glutamine synthase; (2) PDC E1 component, glutamine synthase; (3) PDC E1 and E2 components, RNase, ribosomal proteins L2 and L6; (4) PDC E1 and E2 components, RNase, L2; (5) L2, Obg GTPase, signal recognition particle (SRP) protein; (6) L2, trigger factor, SRP protein (in both tight couple preparations); (7) RNase (in both FLAG 30S preparations). (B) Relative levels of intact L9, L12, and catabolite-controlled protein (CTC) detected by LC–MS. The relative levels of intact proteins L9 and L12 are reported as values normalized to a standard protein added to the sample prior to analysis. CTC levels are represented as percentage intact with respect to detected truncations. The source of the subunits (either centrifugation or FLAG chromatography to give free/loose/tight 50S) and whether a cysteine–Sulfolink precolumn was employed are indicated on the x axis. The seven open bars on the left of the histograms are values from a selection of subunits purified by centrifugation. These are presented for comparison with the subunits obtained from chromatography.

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dissociation and CTC being subject to multiple C-terminal cleavages (McLellan et al., submitted for publication). Relative levels of L9, L12, and intact CTC are compared with seven 50S preparations made by centrifuge in Fig. 4. Levels of the proteins are generally higher for 50S purified from 70S by FLAG chromatography, with a slight further improvement using the precolumn. For the free 50S subunits, which do not bind the FLAG column, contents of L9 and L12 were low but were increased by the precolumn.

35

25 20 15

14

C cpm X 10 3

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5 0 50S source: Centrifugation

_ _ _

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Free

_ +

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_

+

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_

+

+ +

Fig. 5. Activity of 50S preparations (together with excess 30S) in polyU-directed synthesis of polyPhe in vitro. The source of the 50S subunits (either centrifugation or FLAG chromatography to give free/loose/tight 50S) and whether a cysteine– Sulfolink precolumn was employed are indicated on the x axis.

those derived from tight couples are nearly as pure (lanes 7 and 8), as are the 30S subunits (lanes 9 and 10). In-gel tryptic digests of the high-molecular-weight contaminant bands (numbered on Fig. 4) were subjected to LC–MS/MS analysis. A full listing is given in the Fig. 4 legend. Components E1 and E2 of the pyruvate dehydrogenase complex (PDC) were found in all four bands in the free 50S fraction from the centrifuged column flow-through (lane 3). The presence of PDC could be anticipated because this sample was isolated by sucrose density gradient, where the complex is known to cosediment with the 50S subunit [27]. Similarly, the presence of glutamine synthase in this sample is likely due to its large size (800 kDa) and perhaps its tendency to associate with nucleic acids [28]. An RNase was also detected in this sample and in both 30S samples. Protein L6 was found migrating at approximately 40 kDa in this 50S sample, and protein L2 was found migrating at approximately 50 to 55 kDa in many 50S preparations along with signal recognition particle (SRP) protein. Obg guanine nucleoside triphosphatase (GTPase) and trigger factor were also found. We routinely subject ribosomal proteins extracted from D. radiodurans 50S preparations to LC–MS and compare contents of individual proteins identified on the LC absorbance trace using their mass spectrum. Relative contents of L9, L12, and catabolitecontrolled protein (CTC, an L25 homolog) vary in 50S preparations made by centrifugation, with proteins L9 and L12 being prone to

1.5

A A

HAT-tagged 30S HAT-tagged 30S subunits were purified by chromatography on an Ni–NTA column (see Materials and methods). The HAT-tagged sample sedimented similarly to the FLAG-tagged 30S (Fig. 2E) on sucrose density gradients (Fig. 6A). The rRNA and proteins extracted from this sample are displayed in Figs. 3 and 4 (lane 11 in both). This material contains 30S ribosomal proteins but also many contaminating proteins, confirming the original premise that direct binding and elution of subunits can result in impurities. The use of a cysteine–Sulfolink precolumn allowed cleaner 30S subunits to be obtained from the Ni–NTA column in a similar manner to that observed with the FLAG purification (not shown). Binding of the 70S couples to the resin was unexpectedly low. The sucrose density gradient profiles before and after binding of lysate to the column (Fig. 6B) show that most of the 30S subunits, but only some of the 70S couples, have bound. (Note that most of the polysomes have pelleted on these linear gradients.) The material sedimenting between the 50S and 70S are loose couples that dissociate into subunits if centrifuged at the lower magnesium concentration of 6 mM, as seen in other studies [13]. After passage 100

0.75

30S 30S

50S subunits were incubated with 30S subunits in polyU-directed polyPhe synthesis assays in vitro to determine their synthetic activity. (The 30S subunits used had been purified by FLAG chromatography because these had slightly better activity than 30S from centrifugation.) 50S subunits purified by FLAG chromatography alone had similar activity to those made by centrifuge alone (Fig. 5), and activities of both preparations were improved dramatically by the cysteine–Sulfolink precolumn. Activity of the free 50S fraction was very low even with the precolumn. We have found that the integrity of 23S rRNA correlates well with in vitro activity for 50S from D. radiodurans so that the most active subunits here are those with the most intact rRNA (Fig. 3 and McLellan et al., submitted for publication).

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Fig. 6. Purification of HAT-tagged 30S subunits. (A) Sucrose density gradient profile of 30S subunits eluted from an Ni–NTA column after binding of a HAT–S16 cell lysate. An asterisk (*) marks a peak of 30S dimers. (B) Overlaid sucrose density gradient absorbance profiles of the cell lysate before (black line) and after (gray line) passage through the column. (C) Binding of HAT-tagged 30S subunits to Ni–NTA resin on their own (open circles) or in 70S ribosomes (closed circles).

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through the column, some unidentified material is seen sedimenting a little slower than 30S. This might indicate enrichment of an unfolded 30S fraction that fails to bind or a detrimental effect of passage through the resin such as sensitivity to the nickel ions that can leach from such resins. Although a few 50S subunits could be eluted from the column at low magnesium, yields were unacceptably low and the use of different buffers, or cobalt rather than nickel resin, did not improve the yields (see Materials and Methods). Further analysis was performed by purifying half of the 70S ribosomes from the unbound fraction on sucrose density gradients made in lysis buffer and half on gradients made in low magnesium buffer to isolate 30S from the same 70S. Analytical sucrose density gradients of the resulting samples showed that the 30S sample was pure but that the 70S sample contained 14% contamination by free 50S subunits. Binding of the 30S and 70S to resin was compared by batch binding, with a correction being made for this contamination. Binding of the 30S is much more efficient than that of the 70S (Fig. 6C) despite the fact that such a problem was not encountered with the FLAG-tagged variant. It may be that a conformational change in the 30S on association with the 50S in a 70S couple favors sequestration of the HAT tag so that it is not accessible to the resin.

Discussion Purification of 50S subunits by association with FLAG-tagged 30S yielded highly pure preparations of subunits on a large scale. As with ribosomes purified from D. radiodurans by centrifugation, there was some degradation of rRNA, but protein integrity and content were improved. Activity in polyU-directed in vitro translation was comparable between the two methods. Unlike laboratory strains of E. coli, lysates made from D. radiodurans readily degrade rRNA and proteins and its ribosomes are prone to loss of activity during purification. Comparison of the cysteine–Sulfolink and FLAG chromatographies shows that cysteine–Sulfolink is especially effective at preventing this degradation. During the preparation of this article, a method for His-tagmediated purification of ribosomes from E. coli was published [29]. The method tags the C terminus of protein L12 and so is analogous to the C-terminal tagging of its homologs (P1 and P2) in yeast [15]. Our expectation that tagged stalk proteins might prove to be an unreliable handle for capture is proved to be incorrect, as the method captures 70S ribosomes efficiently. The authors attribute the success of L12 tagging compared with less successful His tagging of some other proteins tested (e.g., S2, S3, L30) to the four copies of L12 in the 50S subunit. However, given that a single copy of the weaker binding FLAG tag has been successful in both Saccharomyces cerevisiae and D. radiodurans, the success may in fact be due to reduced interaction of the His tag on L12 with rRNA. The C-terminal domains of the stalk are thought to populate two states: one that extends away from the subunit surface and one where the proteins fold back and interact with the subunit surface [30]. Interestingly, free 50S subunits were not recovered in the elution. If this proves to be a reversal of our experience with HATtagged S16 where only free 30S, and not 70S, could be captured, then it may be that the stalk spends more time folded down onto the 50S surface in the free subunit than in the 70S couple so that 70S, but not free 50S, can be captured. Our method is gentle enough that some common ribosomeassociated proteins were recovered; a simple salt wash could likely remove these associated proteins if required. For instance, the associated proteins included those that interact with the emerging nascent peptide such as SRP protein and trigger factor [31]; these could be removed by sedimentation through 1.1 M sucrose in buffer containing 0.5 M NH4Cl (see Materials and methods). The ObgG

protein seen in free 50S preparations is implicated both in ribosome assembly and in the stringent response to amino acid starvation [27,32]. RelA, another stringent response protein known to associate with ribosomes, eluted with the 30S, although association with the 50S would have been expected [33]. LC–MS of ribosomal proteins of the D. radiodurans 50S subunit (see Ref. [34] and McLellan et al., submitted for publication) has shown that the observed masses of proteins L2 and L6 match their sequence masses (29.9 and 19.4 kDa, respectively). Therefore, the observation of forms of ribosomal proteins L2 and L6 that migrate significantly above 36 kDa on reducing SDS–PAGE was unexpected and may reflect formation of covalent complexes that include protein L2 or L6. The separate isolation of loosely and tightly associated 50S is an additional consequence of the strategy. Loose and tight 70S couples are conformers; tight couples are more compact [35] and differ in structure at sites that interact with elongation factors (EFs) such as the translocation factor EF-G [36–39]. Loose couple ribosomes likely correspond to the ratcheted state of the ribosome, where the 30S subunit is rotated relative to the 50S [40]. Both the loose and ratcheted states are promoted by binding of EF-G with a nonhydrolyzable GTP analog, whereas hydrolysis of the GTP (by EF-G/ GTP/fusidic acid) restores the conformation to a nonratcheted (tight) state [40,41]. Ratcheting is part of an intrinsic mechanism to translocate tRNAs from the classic A and P sites to the hybrid A/P and P/E sites on the ribosome [42]. Because ribosomes from D. radiodurans are subject to degradation by endogenous nucleases and proteases during purification, degradation might be expected to differ for the structurally different 70S conformers. For instance, tight couple 23S rRNA has been reported to be more prone to nuclease action [43]. However, little difference was seen between the integrity of 23S rRNA of subunits purified from loose and tight couples here. Only the nonassociated 50S subunits differed much in the integrity of their RNA and protein, showing greater degradation that is likely due to longer exposure to nucleases and proteases combined with a lack of protection otherwise afforded by association with the 30S subunit. Thus, separation of free and associated 50S improves homogeneity. However, none of the samples could be crystallized under the conditions used to crystallize the 50S purified by centrifugation. In further work (McLellan et al., submitted for publication), we have shown that, even for 50S purified by centrifuge, use of the cysteine–Sulfolink precolumn prevents subsequent crystallization, and it is thought that in such cases the rRNA is actually too intact for crystallization. (The 50S subunits purified by centrifugation that were used for comparison with 50S from chromatography in Figs. 4B and 5 all crystallize.) However, although loose couple 50S purified by FLAG chromatography without the precolumn have purity, integrity, and activity properties well within the range of crystallizable 50S purified by centrifuge, even extensive robotic screening of crystallization conditions failed to yield any crystals of these subunits. This unexpected outcome may hold useful clues as to what does (and does not) potentiate subunit crystallization. Alternatively, it may be that extension of the method to other tags or locations on the ribosome (e.g., a His tag on protein L12 as used in E. coli) could produce subunits that do crystallize. We tested the FLAG tag because it was precedented, but anti-FLAG affinity resin is costly. Although the cheaper HAT tag was unsuccessful on protein S16, there are other cost-effective systems, such as the Strep tag (IBA), that could be worth testing with this protein. We have demonstrated rapid purification of 50S subunits from D. radiodurans by FLAG–30S affinity chromatography on a large scale. The ribosomal subunits have good protein integrity, purity, homogeneity, and activity so that they will be suitable for a range of downstream applications. The use of this purification by subunit association, together with cysteine–Sulfolink chromatography,

Purification of the large ribosomal subunit / S.P. Simons et al. / Anal. Biochem. 395 (2009) 77–85

provides a powerful combination to maximize subunit quality in an efficient and scalable process. Furthermore, the principle of on-column selective dissociation demonstrated here may also be applicable to the purification of components of other macromolecular complexes. Acknowledgment We are indebted to Toshifumi Inada (Nagoya University) for his generous gift of yeast strain YIT-613. References [1] P.C. Jelenc, Rapid purification of highly active ribosomes from Escherichia coli, Anal. Biochem. 105 (1980) 369–374. [2] S.V. Kirillov, V.I. Makhno, N.N. Peshin, Y.P. Semenkov, Separation of ribosomal subunits of Escherichia coli by Sepharose chromatography using reverse salt gradient, Nucleic Acids Res. 5 (1978) 4305–4315. [3] B.A. Maguire, L.M. Wondrack, L.G. Contillo, Z. Xu, A novel chromatography system to isolate active ribosomes from pathogenic bacteria, RNA 14 (2008) 188–195. [4] A.E. Hesslein, V.I. Katunin, M. Beringer, A.B. Kosek, M.V. Rodnina, S.A. Strobel, Exploration of the conserved A + C wobble pair within the ribosomal peptidyl transferase center using affinity purified mutant ribosomes, Nucleic Acids Res. 32 (2004) 3760–3770. [5] A.A. Leonov, P.V. Sergiev, A.A. Bogdanov, R. Brimacombe, O.A. Dontsova, Affinity purification of ribosomes with a lethal G2655C mutation in 23S rRNA that affects the translocation, J. Biol. Chem. 278 (2003) 25664–25670. [6] E.M. Youngman, R. Green, Affinity purification of in vivo-assembled ribosomes for in vitro biochemical analysis, Methods 36 (2005) 305–312. [7] X. Gan, M. Kitakawa, K-I. Yoshino, N. Oshiro, K. Yonezawa, K. Isono, Tagmediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components, Eur. J. Biochem. 269 (2002) 5203–5214. [8] P. Harnpicharnchai, J. Jakovljevic, E. Horsey, T. Miles, J. Roman, M. Rout, D. Meagher, B. Imai, Y. Guo, C.J. Brame, J. Shabanowitz, D.F. Hunt, J.L. Woolford Jr., Composition and functional characterization of yeast 66S ribosome assembly intermediates, Mol. Cell 8 (2001) 505–515. [9] C. Saveanu, M. Fromont-Racine, A. Harington, F. Ricard, A. Namane, A. Jacquier, Identification of 12 new yeast mitochondrial ribosomal proteins including 6 that have no prokaryotic homologues, J. Biol. Chem. 276 (2001) 15861–15867. [10] A. Zikova, A.K. Panigrahi, R.A. Dalley, N. Acestor, A. Anupama, Y. Ogata, P.J. Myler, K. Stuart, Trypanosoma brucei mitochondrial ribosomes: affinity purification and component identification by mass spectrometry, Mol. Cell. Proteomics 7 (2008) 1286–1296. [11] G. Rigaut, A. Shevchenko, B. Rutz, M. Wilm, M. Mann, B. Seraphin, In the laboratory: a generic protein purification method for protein complex characterization and proteome exploration, Nat. Biotechnol. 17 (1999) 1030– 1032. [12] R.S. Zitomer, J.G. Flaks, Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association, J. Mol. Biol. 71 (1972) 263–279. [13] B. Hapke, H. Noll, Structural dynamics of bacterial ribosomes: IV. Classification of ribosomes by subunit interaction, J. Mol. Biol. 105 (1976) 97–109. [14] T. Inada, E. Winstall, S.Z. Tarun Jr., J.R. Yates III, D. Schieltz, A.B. Sachs, One-step affinity purification of the yeast ribosome and its associated proteins and mRNAs, RNA 8 (2002) 948–958. [15] E. Guarinos, C. Santos, A. Sanchez, D-Y. Qiu, M. Remacha, J.P.G. Ballesta, Tagmediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure, Mol. Microbiol. 50 (2003) 703–712. [16] H. Tokimatsu, W.A. Strycharz, A.E. Dahlberg, Gel electrophoretic studies on ribosomal proteins L7/L12 and the Escherichia coli 50 S subunit, J. Mol. Biol. 152 (1981) 397–412. [17] B.T. Wimberly, D.E. Brodersen, W.M. Clemons Jr., R.J. Morgan-Warren, A.P. Carter, C. Vonrhein, T. Hartsch, V. Ramakrishnan, Structure of the 30S ribosomal subunit, Nature (Lond.) 407 (2000) 327–339. [18] J.M. Zengel, L. Lindahl, Diverse mechanisms for regulating ribosomal protein synthesis in Escherichia coli, Prog. Nucleic Acid Res. Mol. Biol. 47 (1994) 331– 370. [19] J.M. Lovgren, P.M. Wikstrom, Hybrid protein between ribosomal protein S16 and RimM of Escherichia coli retains the ribosome maturation function of both proteins, J. Bacteriol. 183 (2001) 5352–5357.

85

[20] R. Meima, M.E. Lidstrom, Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli–D. radiodurans shuttle vectors, Appl. Environ. Microbiol. 66 (2000) 3856–3867. [21] P.D. Gutman, P. Fuchs, L. Ouyang, K.W. Minton, Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans, J. Bacteriol. 175 (1993) 3581–3590. [22] S. Tirgari, B.E.B. Moseley, Transformation in Micrococcus radiodurans: measurement of various parameters and evidence for multiple, independently segregating genomes per cell, J. Gen. Microbiol. 119 (1980) 287–296. [23] K.S. Udupa, P.A. O’Cain, V. Mattimore, J.R. Battista, Novel ionizing radiationsensitive mutants of Deinococcus radiodurans, J. Bacteriol. 176 (1994) 7439– 7446. [24] S. Petry, D.E. Brodersen, F.V.I.V. Murphy, C.M. Dunham, M. Selmer, M.J. Tarry, A.C. Kelley, V. Ramakrishnan, Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon, Cell 123 (2005) 1255–1266. [25] M. Wilm, A. Shevchenko, T. Houthaeve, S. Breit, L. Schweigerer, T. Fotsis, M. Mann, Femtomole sequencing of proteins from polyacrylamide gels by nanoelectrospray mass spectrometry, Nature (Lond.) 379 (1996) 466–469. [26] C. Pande, A. Wishnia, Pressure dependence of equilibria and kinetics of Escherichia coli ribosomal subunit association, J. Biol. Chem. 261 (1986) 6272– 6278. [27] M. Jiang, K. Datta, A. Walker, J. Strahler, P. Bagamasbad, P.C. Andrews, J.R. Maddock, The Escherichia coli GTPase CgtAE is involved in late steps of large ribosome assembly, J. Bacteriol. 188 (2006) 6757–6770. [28] S.L. Streicher, B. Tyler, Purification of glutamine synthetase from a variety of bacteria, J. Bacteriol. 142 (1980) 69–78. [29] J. Ederth, C.S. Mandava, S. Dasgupta, S. Sanyal, A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli, Nucleic Acids Res. 37 (2009) e15. [30] F.A.A. Mulder, L. Bouakaz, A. Lundell, M. Venkataramana, A. Liljas, M. Akke, S. Sanyal, Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome, Biochemistry 43 (2004) 5930–5936. [31] R.S. Ullers, E.N.G. Houben, J. Brunner, B. Oudega, N. Harms, J. Luirink, Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle, J. Biol. Chem. 281 (2006) 13999– 14005. [32] P. Wout, K. Pu, S.M. Sullivan, V. Reese, S. Zhou, B. Lin, J.R. Maddock, The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase, J. Bacteriol. 186 (2004) 5249–5257. [33] S. Ramagopal, B.D. Davis, Localization of the stringent protein of Escherichia coli on the 50S ribosomal subunit, Proc. Natl. Acad. Sci. USA 71 (1974) 820– 824. [34] W.E. Running, J.P. Reilly, Ribosomal proteins of Deinococcus radiodurans: their solvent accessibility and reactivity, J. Proteome Res. 8 (2009) 1228–1246. [35] A. Bonincontro, K.H. Nierhaus, G. Onori, G. Risuleo, Intrinsic structural differences between ‘‘tight couples” and Kaltschmidt–Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry, FEBS Lett. 490 (2001) 93–96. [36] D. Dash, F.E. Abidi, D.P. Burma, Fluorescence anisotropy of IAEDANS-labelled L7/L12 attached to rRNAs and ribosomes, Indian J. Biochem. Biophys. 25 (1988) 226–229. [37] D. Dash, S. Mahanti, D.P. Burma, Conformational change of L7/L12 stalk in the different functional states of 50S ribosomes, J. Biosci. 11 (1987) 561– 569. [38] D.N. Dey, D.P. Burma, Polyclonal antibodies as probes to distinguish between tight and loose couple 50S ribosomes of Escherichia coli, Indian J. Biochem. Biophys. 28 (1991) 369–373. [39] R. Morishita, S. Yoshinari, Y. Endo, Probing the structure of a-sarcin/ricin domain in Escherichia coli ribosomes, Nucleic Acids Symp. Ser. 35 (1996) 291– 292. [40] J. Frank, R.K. Agrawal, A ratchet-like inter-subunit reorganization of the ribosome during translocation, Nature (Lond.) 406 (2000) 318–321. [41] D.P. Burma, A.K. Srivastava, S. Srivastava, D. Dash, Interconversion of tight and loose couple 50S ribosomes and translocation in protein synthesis, J. Biol. Chem. 260 (1985) 10517–10525. [42] P.V. Cornish, D.N. Ermolenko, H.F. Noller, T. Ha, Spontaneous intersubunit rotation in single ribosomes, Mol. Cell 30 (2008) 578–588. [43] D.P. Burma, A.K. Srivastava, S. Srivastava, D.S. Tewari, D. Dash, S.K. Sengupta, Differences in physical and biological properties of 50S ribosomes and 23S RNAs derived from tight and loose couple 70S ribosomes, Biochem. Biophys. Res. Commun. 124 (1984) 970–978.