Technical Paper
RNA Biology 7:3, 354-360; May/June 2010; © 2010 Landes Bioscience
Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method Jonathan A. Leshin,1 Rasa Rakauskaitė,1 Jonathan D. Dinman1,* and Arturas Meskauskas1,2,* Department of Cell Biology & Molecular Genetics; University of Maryland; College Park, MD USA; 2Department of Biotechnology and Microbiology; Vilnius University; Vilnius, Lithuania
1
Key words: ribosome, purification, yeast, rRNA, protein, chromatography, tRNA
One of the major challenges facing researchers working with eukaryotic ribosomes lies in their lability relative to their eubacterial and archael counterparts. In particular, lysis of cells and purification of eukaryotic ribosomes by conventional differential ultracentrifugation methods exposes them for long periods of time to a wide range of co-purifying proteases and nucleases, negatively impacting their structural integrity and functionality. A chromatographic method using a cysteine charged Sulfolink resin was adapted to address these problems. This fast and simple method significantly reduces co-purifying proteolytic and nucleolytic activities, producing good yields of highly biochemically active yeast ribosomes with fewer nicks in their rRNAs. In particular, the chromatographic purification protocol significantly improved the quality of ribosomes isolated from mutant cells. This method is likely applicable to mammalian ribosomes as well. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.
Introduction With the elucidation of atomic resolution of ribosome structures,1-4 and of near atomic resolution cryo-EM studies (reviewed in ref. 5), the past decade has witnessed a dramatic resurgence of interest in the ribosome. The eukaryotic ribosome has particularly emerged as an area of intense focus (reviewed in ref. 6). With its superior molecular genetics and biochemical toolset, the yeast ribosome is highly amenable to addressing many of the pressing questions in the field. However, the larger size of eukaryotic ribosomes, their much more complex biogenesis programmes, and in particular, the release of cellular proteases and nucleases concomitant with cellular disruption impose considerable barriers to researchers in the field, particularly with regard to structure/function analyses. Ribosome purification protocols have differed little from the original differential centrifugation methods pioneered over half a century ago.7 This essentially involves lysing cells, harvesting a cytosolic fraction from a low speed spin, and then pelleting ribosomes by high speed centrifugation. Although additional steps have been added along the years, e.g., salt washes, and glycerol cushions, biochemical and structural studies of yeast ribosomes have been hampered by their tendency to become degraded during the purification process, most likely due to the long periods of time during the ultracentrifugation steps during which ribosomes are exposed to these classes of enzymes.8
Column chromatography based methods could potentially enhance the rates at which ribosomes could be separated from contaminants. Tagged ribosomal proteins expressed in vivo have been used to affinity purify yeast ribosomes, although these have not been characterized biochemically or structurally.9 Unlike their bacterial counterparts, efforts to purify yeast ribosomes by inserting RNA-based affinity tags into rRNAs have not yet been successful, although one such effort resulted in the discovery of the nonfunctional rRNA decay pathway.10 The high levels of proteases and nucleases present in clinical isolates of pathogenic bacteria led Maguire and co-workers to devise a chromatographic method for ribosome purification using a cysteine charged Sulfolink resin.11 The rRNAs and proteins derived from bacterial ribosomes isolated using this method showed much lower levels of degradation, and the ribosomes so purified were significantly more able to bind erythromycin and to synthesize proteins. These observations suggested that the cysteine charged sulfolink resin chromatography method may also be applicable to yeast ribosomes, and if so, that it could solve many of the problems described above. Here, we show that this is indeed the case: the column chromatographic method rapidly and efficiently results in dissociation of a significant fraction of contaminating nucleases and proteases from ribosomes resulting in purer, more biochemically active ribosome preparations with enhanced biochemical and structural properties, and that scales well to higher quantities
*Correspondence to: Jonathan Dinman/ Arturas Meskauskas; Email:
[email protected]/
[email protected] Submitted: 02/01/10; Revised: 02/20/10; Accepted: 02/22/10 Previously published online: www.landesbioscience.com/journals/rnabiology/article/11648 354
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Technical Paper
Technical Paper
ribosomes (CS), ribosomes purified after ultracentrifugation through the glycerol cushion (CR), the glycerol cushion/supernatant of the overnight spin of traditional method (TS), and ribosomes purified using the traditional method after ultracentrifugation through the glycerol cushion (TR). RNAs were extracted from each aliquot and separated through glyoxal denaturing agarose gels, with 1 µg of each sample used. A sample gel is shown in Figure 2A. In the initial S30 phase, 25S, 18S rRNAs as well as tRNAs are present and remain so until after the first wash. The presence of rRNA in the flowthrough and first wash steps indicates that there is some loss of ribosomes, likely due to saturation of the resin beyond its binding capacity. The second and third washes contain little rRNA and decreasing amounts of tRNA, indicating that very few ribosomes are lost after the first wash. The presence of both the rRNAs and tRNA in the elution fraction demonstrates that the resin binds both types of RNA. The high level of tRNA in this fraction indicates that the resin binds tRNA well, which may prevent optimal ribosome binding. Additionally, some tRNA contamination present in the elution fraction is due to the presence of tRNA inside the ribosomes. Overnight high speed centrifugation of samples through a glycerol cushion removed all of the remaining tRNAs from the samples (CS), an important consideration for many downstream biochemical assays, e.g., ribosome/ligand binding or rRNA structural analyses (see below). Purified ribosomes (CR) lacked tRNAs. Agarose gel analysis of RNAs extracted from ribosomes using the traditional method showed no detectable tRNAs. The TS fraction contains a small amount of rRNA as well as tRNA. Figure 1. Methods flowchart. Ribosome purification by the “traditional” method The effects of the column purification method was is shown at left. Chromatographic purification using the cysteine linked sulfolink also monitored at the protein level using aliquots taken resin is depicted to the right. from the above fractions. Proteins from all fractions were denatured and resolved through a SDS-PAGE gel (Fig. of ribosomes. This method adds a significant new tool to the 2B). Similar to the RNA analyses, the S30 and flowthrough fractions from the chromatographic preparations contained hetarsenal of ribosomologists. erogeneous mixtures of cellular proteins, and the wash fractions showed decreasing amounts of proteins. The elution fraction Results and Discussion (E) and final ribosome fraction (CR) were distinctly different. Our experience working with yeast ribosomes, particularly bio- Specifically, the glycerol cushion supernate contained a hetchemical and structural characterization of those expressing erogeneous mixture of cellular proteins, while the final chrorRNA or ribosomal protein mutants, revealed a pressing need to matographically purified ribosomal fraction contained very few devise a new method to maximize ribosome integrity and func- proteins with molecular weights >50 kDa, consistent with the tion by minimizing the impact of co-purifying nucleolytic and relatively small sizes of ribosomal proteins. The pattern of proproteolytic activities. These concerns led us to investigate a chro- teins present in this sample also matched the pattern observed in matographic protocol employing a cysteine charged sulfolink the traditional ribosome fraction (TR). Quantitative analyses of ribosome purity: RNase and proresin previously used for purifying bacterial ribosomes from clinical isolates.11 A flowchart of the traditional and chromatographic tease assays. Contamination of samples by catabolic enzymes is methods is shown in Figure 1. a major concern in purifying RNAs and proteins. To examine To qualitatively monitor the purification process, aliquots the purity of the different samples, RNase and protease activities were taken from the following steps: S30 spin (S30), flowthrough were monitored using commercially available kits. The RNase after second binding to the resin (F), each wash step (W1-3), activity kit determines the presence of RNase by liberating samples eluted from the resin (E), the glycerol cushion/super- a fluor and a quencher from an RNA substrate, thus allowing natant of the overnight spin of the chromatographically purified detection of the fluor. One µg of protein from each sample was
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Figure 2. (A) RNA gel electrophoresis analyses. Each lane represents a step in the chromatographic purification process. S30, Spin at 30,000 g; F, Flowthrough; W1, 1st wash step; W2, 2nd wash step; W3, 3rd wash step; E, Elution from resin; CS, Glycerol cushion/supernatant fraction from the overnight spin of the chromatographic purification, CR, final ribosome containing fraction from the chromatographic purification; TS, Glycerol cushion/supernatant fraction from traditional purification; TR, final ribosome containing fraction from the traditional purification. All fractions except CR and TR contain tRNA. One µg of RNA was used for each lane. (B) SDS-PAGE analyses. Each lane represents a step in either the chromatographic or traditional purification process. M, Marker; S30, Spin at 30,000 g; F, Flowthrough from chromatographic purification; W1, 1st wash step; W2, 2nd wash step; W3, 3rd wash step; E, Elution from the cysteine charged sulfolink resin; CS, Glycerol cushion/supernatant fraction from the overnight spin of the chromatographic purification; CR, final ribosome containing fraction from the chromatographic purification; TS, Glycerol cushion/supernatant fraction from traditional purification; TR, final ribosome containing fraction from the traditional purification. (C) RNase detection assay. A fluor-quench labeled RNA substrate was added to 1 µg of each fraction. Activity was determined by detection of free fluorophore. (D) Protease Activity Assay. A fluor-labeled casein was added to 5 µg of each fraction. Activity was determined by detection of unquenched fluorescein. Activity is represented per µg.
used in the assay. The chromatographic flowthrough fraction and the glycerol cushion/supernatant fraction derived from the traditional method exhibited the highest levels of RNase activity
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(Fig. 2C). The flowthrough fraction was especially enriched in RNase activity, indicating that RNases do not bind efficiently to the resin and that as ribosomal proteins are removed from the
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mix, nucleases makes up a greater percentage of the remaining proteins. The S30 fraction also contained a slightly lower but significant nuclease activity, consistent with prior observations of high RNase activity in this fraction. Each subsequent wash fraction contained decreasing amounts of RNase activity. Similar amounts of RNase activity were observed in ribosomes purified using “traditional” ultracentrifugation method, and in both the column elution fraction, and the glycerol cushion/supernatant fraction from the chromatographic samples. The lowest level of RNAse activity was observed in the chromatographically purified ribosomes. In the protease assay, degradation of fluorescently labeled casein results in increased FRET homotransfer from the released fluorescein, detected as an increase in RFU. These assays revealed an identical pattern observed in the RNase activity assays (Fig. 2D). Specifically, the column flowthrough and the traditional cushion/supernatant contained the highest protease activities, followed by the S30. This again shows how the removal of ribosomes during the binding to the column enriches the protease content of the flowthrough fraction. Similarly, the cushion/ supernatant portion from the traditional purification method captured a large portion of proteases. Protease activity progressively decreased with each wash fraction. The elution fraction, column-derived cushion/supernatant fraction and traditional ribosomes overlapped, while the chromatographically purified ribosomes contained the lowest protease activity. The cysteine charged Sulfolink resin is very inefficient at binding non-RNA substrates and as such, the proteases and RNases retained beyond the wash steps are likely to be ribosome-associated. The majority of these contaminants were removed during the final overnight ultracentrifugtion step of the purification protocol. Biochemical and structural analyses of purified ribosomes. Minimization of protease and nuclease activities should improve the biochemical and structural properties of purified ribosomes. Three different assays were employed to examine these parameters. Ribosomes have three tRNA binding sites, the A-, P- and E-sites, and saturation experiments have demonstrated that deacylated tRNA Phe binds to polyU programmed ribosomes with the highest affinity at the P-site, followed by the E-site, and then the A-site.12,13 The percentage of active (tRNA binding competent) ribosomes can be used to evaluate the quality of ribosome preparation. To compare the quality of ribosomes isolated using the chromatographic and traditional methods, saturation binding experiments using [32P]tRNA Phe and polyU programmed ribosomes were carried out. The chromatographically purified ribosomes bound ∼2.5 tRNAs/ribosome (87% active ribosomes) as compared to 1.9 tRNAs/ribosome using the traditional method (63% active ribosomes) (Fig. 3A). These indicate that a greater fraction of ribosomes remained functionally competent through the chromatographic purification protocol as compared to the traditional method. Importantly, as noted above, tRNAs species were not detected in total RNA extracts of traditionally purified ribosomes, and thus the apparent saturation at ∼2 tRNAs/ribosome in these preparations is not due to the presence of co-purifying tRNAs. The occupation of all three binding sites was confirmed by the inability of Ac-Phe-tRNA Phe to
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bind to ribosomes that were pre-saturated with deacylated tRNA (Fig. 3B). These polyU programmed ribosomes alone were approximately 83% active for binding Ac-Phe-tRNA Phe. Binding of charged tRNA ([14C]Phe-tRNA Phe) to polyU programmed ribosomes in the presence of exogenously added eukaryotic elongation factors was used to compare the specific activities of ribosomes purified using the two methods. Figure 3C shows that the fraction of active ribosomes, i.e., those able to specifically bind aa-tRNA to the A-site was dramatically enhanced by the chromatographic purification protocol. Specifically, ∼75% of the chromatographically purified ribosomes retained their activity, as compared to ∼15% of ribosomes purified using the traditional method. This is particularly important with regard to ribosomes synthesized in strain JD1458, where ribosomes are expressed from a high copy episomal plasmid. It has been observed that this results in formation of a few small “micro-nucleolar” bodies instead of one large nucleolus.14 This strain is inherently temperature sensitive,15 and we observe that these cells contain significantly more vacuoles (containing nucleases and proteases) and altered morphologies than the parental strain expressing rRNAs from chromosomal rDNA loci (unpublished results). We hypothesize that episomal expression of rRNAs somehow interferes with optimal ribosome biogenesis, increasing the amount of ribosome turnover through the non-functional ribosome pathway.16 In fact, we have noticed that this is appears to be a general problem in working with yeast cells expressing mutant ribosomes. The observation that the chromatographic method results in a dramatic increase in active ribosomes isolated from this strain provides us with a powerful tool for high quality biochemical analyses of mutant ribosomes. The lower levels of nucleases and proteases (Fig. 2C and D), and the enhanced tRNA binding properties of the chromatographically purified ribosomes suggested that these were more structurally intact than their traditionally purified counterparts. To assay this, 25S rRNA extracted from the two purified preparations were directly sequenced by reverse transcriptase primer extension. Sequencing reactions from the traditionally purified ribosomes contained much higher levels of background noise, i.e., faint bands across all lanes, as compared to those from chromatographically purified ribosomes (Fig. 3D). These background bands, i.e., strong stops, are typically due to either the presence of strong secondary RNA structures (which impede the progress of the reverse transcriptase), or to the accumulation of single-strand breaks in the rRNA. Again, these ribosomes were isolated from strain JD1458, where we have observed high levels of background bands due to rRNA nicking. The near total lack of strong stops in the chromatographically purified samples indicates that the large amount of strong stops in the traditional samples are due to higher levels of rRNA degradation in these samples, consistent with the higher levels of RNase contamination of the traditionally purified ribosomes. Summary. The presence of large amounts of co-purifying nucleases and proteases has hampered biochemical and structural characterization of eukaryotic ribosomes, particularly those isolated from yeast. Here, we describe an adaptation of a chromatographic purification method to solve this problem.
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Yeast ribosomes isolated using this method are much more biochemically active and their RNAs significantly less nicked than those isolated using the traditional method. We also suggest that this protocol should also be applicable to purification of ribosomes from higher eukaryotes, including human cells. Materials and Methods Yeast strains and reagents. Yeast strain JD932 (MATa ade2-1 trp1-1 ura3-1 leu2-3,112 his3-11,15 can1-100) was used for the protease and RNase activity assays. Yeast Strain JD1370 (MATa trp1 ura3 leu2 PEP4::HIS3 NUC1::LEU2) was used for the agarose and acrylamide gels and the [32P]tRNA Phe and Ac-[14C]Phe-tRNA Phe binding assays. Strain JD1458 [MATa ade2-1 ura3-1 trp1-1 his3-11 leu2-3, 112 can1-100 ∆rDNA::his3::hisG + pJD180.TRP (pPol I-35S rDNA, 5S rDNA, TRP1, 2 µ, ampr)] was used to monitor binding of [14C]Phe-tRNA Phe to ribosomal A-sites and for reverse transcriptase sequencing of rRNA. Yeast were grown in YPAD to an OD595 of between 0.8–1.5 units. Sulfolink and 10 ml centrifuge columns were purchased from Pierce (Rockford, IL). Chemicals used were purchased from Sigma-Aldrich (St. Louis, MO), Invitrogen (Carlsbad, CA) and Bio-Rad (Hercules, CA). Preparation of a cysteine charged sulfolink resin. Resin was prepared at room temperature. A total of 10 ml of a 50% slurry of Sulfolink coupling gel was placed in two 10 ml plastic vials, with 5 ml distributed into each vial. Vials were centrifuged briefly at 850 xg and supernatants were carefully removed. The gel was washed three times in coupling bufFigure 3. tRNA binding activities and rRNA analysis of ribosomes purified using traditional and chromatographic methods. (A) tRNA saturation binding curves. Ribosomes purified usfer (50 mM Tris, pH 8.5, 5 mM EDTA) by ing chromatographic and traditional methods were programmed with PolyU and incubated resuspending the beads in 5 ml, centrifuging with increasing amounts of deacylated [32P] labeled tRNAPhe. Y-axis denotes the fraction briefly at 850 xg and removing the supernaof tRNAs bound per ribosome. X-axis indicates the ratio of input tRNAs to ribosomes. (B) tant by pipette. Five ml of a 50 mM solution Competition experiment. PolyU programmed, chromatographically purified ribosomes were of L-cysteine, 50 mM Tris, pH 8.5, 5 mM either first incubated with saturating quantities (500 nM of 80S ribosomes with tRNAPhe in 25-fold excess) of unlabeled deacylated tRNAPhe (+tRNA), or buffer alone, and then incubated EDTA buffer was added to each tube and the with 3-fold molar excess of Ac-[14C]Phe-tRNAPhe. The ratios of Ac-[14C]Phe-tRNAPhe bound slurry was mixed for 1 hour at 25°C. Residual per ribosome are indicated. (C) Single site binding isotherms of [14C]Phe-tRNA to A-sites of L-cysteine was removed by washing as above. ribosomes purified from JD1458 (episomally expressed rRNAs) using chromatographic and The gel was resuspended in 10 ml of bindtraditional methods. (D) Direct reverse transcriptase sequencing of 25S rRNA expressed in ing buffer (10 mM Tris-HCl, pH 7.5, 10 mM JD1458 extracted from ribosomes purified using chromatographic (C) and traditional (T) methods. Read shows sequence from G2945 (bottom) to G2848 (top). MgCl2, 60 mM NH4Cl, 2 mM DTT), and the resin equilibrated by washing in 10 ml of binding buffer 3 times as above. The resin was stored in 10 ml of Chromatographic purification of ribosomes using a cysteine binding buffer. The resin capacity was determined to be ∼20 A 260 charged sulfolink resin. As much as possible, all components units per ml. Resin was decanted into a 10 ml centrifuge column, were maintained on ice and all solutions were prepared using capped and stored at 4°C. Two ml of resin were used for every RNase-free water and sterile filtration. One gram of yeast cells one gram of cells. were washed in binding buffer and centrifuged at 3,700 xg for
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5 minutes at 4°C. Cells were resuspended in 1 ml of binding buffer to an approximate total volume of 2 ml, and disrupted using glass beads chilled to 4°C using a Biospec Mini-bead beater (Bartlesville, OK). Unbroken cells, organelles, and cellular debris were removed by centrifugation at 30,000 xg for 30 minutes in a Beckman-Coulter Optima Max E ultracentrifuge (Fullerton, CA). Immediately before use, the resin was pelletted for one minute at 1,000 xg to remove the storage solution. Supernatants from the 30,000 xg spin were removed, taking care to minimize contamination from either the lipid fraction at the very top or the cell debris at the bottom of the tubes, and placed directly into the charged Sulfolink slurry. These mixtures were incubated on ice for 15 minutes. The columns were placed into 15 ml conical tubes and centrifuged at 1,000 xg for one minute. The flowthrough fractions were placed back into the tubes, mixed by hand, and incubated for a further 15 minutes. Columns were capped and 5 ml of binding buffer was added, the columns were mixed by hand until resuspended, the caps removed, and columns centrifuged at 1,000 xg for one additional minute. After repeating this washing protocol two more times, 1.5 ml of elution buffer (10 mM Tris-HCl, pH 7.5, 10 mM Mg2Cl, 500 mM KCl, 2 mM DTT, 0.5 mg/ml heparin) was added to each column. Slurries were mixed by hand, incubated on ice for 2 minutes, columns were placed into new 15 ml conical tubes, and centrifuged at 1,000 xg for 1 minute. This final elution step was repeated once more so that the final sample volumes were ∼3 ml. Used resin was washed with binding buffer without heparin, and stored in 10 ml volumes at 4°C for re-use later. Ribosome containing samples were further purified through glycerol cushions as follows. One ml of buffer C (50 mM HEPES, pH 7.6, 5 mM Mg(CH3COO)2, 50 mM NH4Cl, 1 mM DTT, 25% glycerol) was placed into a polycarbonate ultracentrifuge tube, ribosome containing elution fractions were gently layered on top of 3 ml buffer C, and samples were centrifuged at 100,000 xg overnight. Supernatants were aspirated and pellets containing purified ribosomes were twice washed gently with 1 ml of buffer C. Ribosomes were resuspended in 100 µl of buffer C by gentle disruption using a glass rod. After disruption, tubes were covered with parafilm and shaken at a moderate speed in a cold room vortex for 30 minutes. The contents were transferred to a microcentrifuge tube, centrifuged for 5 minutes at maximum speed, and supernatants were removed to fresh tubes. The purified ribosomes were spectrophotometrically quantified (1 A 260 = 20 pmoles of yeast ribosomes), and stored at -80°C. Purification of ribosomes by differential ultracentrifugation: the “traditional” method. Ribosomes were purified by differential ultracentrifugation as previously described.17 Briefly, yeast cells were suspended in binding buffer with 0.5 mg/ml heparin and disrupted using glass beads in a Biospec Mini-bead beater. Cytosolic fractions from the cell lysates were obtained by centrifugation for 30 min at 30,000 xg, 3 ml of which was layered onto 1 ml of buffer C and ribosomes were pelleted by overnight centrifugation at 100,000 xg. Supernatants were aspirated, pellets were washed twice with buffer C, and ribosomes were resuspended in 200 µl of buffer C using a glass rod to gently disrupt the pellet. The solutions were mixed gently for 30 minutes
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at 4°C, transferred to microcentrifuge tubes and centrifuged at maximum speed for 5 minutes. The supernatant was diluted to 3 ml using elution buffer (containing 500 mM KCl, i.e., salt washed ribosomes), layered over 1 ml of buffer C + 25% glycerol and centrifuged at 300,000 xg for 4 hours. Supernatants were aspirated, pellets washed twice with buffer C and resuspended in 200 µl of buffer C. The solutions were again mixed gently for 30 minutes in a cold room, transferred to microcentrifuge tubes and centrifuged at maximum speed for 5 minutes. The purified ribosomes were spectrophotometrically quantified and stored at -80°C as described above. Electrophoretic separation of RNAs and proteins. RNAs were extracted with phenol/chloroform and spectrophometrically quantified using a Nanodrop 1000 (Wilmington, DE). Glyoxal denatured RNA samples, 1 µg each, were separated through 1% agarose gels, stained with ethidium bromide and visualized under ultraviolet light. A 0.24–9.5 kb ladder (Invitrogen, Carlsbad, CA) was used to calibrate nucleic acid lengths. Protein concentrations were measured using a Quick Start Bradford kit (Bio-Rad). For each sample, 5 µg of protein were separated through 4.8% –12% SDS-PAGE, and visualized with GelCode Blue Safe Protein Stain (Pierce, Rockford, IL). Fermentas Spectre Multicolor Broad Range Protein Ladder (Glen Burnie, MD) was used to calibrate molecular weights. RNase and protease assays. An RNase Alert Test Kit (Ambion, Austin, TX) was used to determine RNase activity, using the instructions provided. Briefly, 5 µl of 10X RNase Alert Lab Test Buffer was added to each tube of fluorescent substrate. One µg of each sample to be tested was added to each tube and the sample was brought to 50 µl using RNase free water. RNase free water, and 5 µl of RNase A in 40 µl of RNase free water were used as positive and negative controls respectively. The samples were loaded into a 96 well plate, and fluorescence activities (excitation 490 nm/emission 520 nm) was read using a Bio-Tek Synergy HT (Winooski, VT), with readings taken every 5 minutes for 1 hour at 37°C. A Pierce Fluorescent Protease Assay Kit (Rockford, IL) was used to determine protease activity, using the instructions provided. Briefly, 5 µg of protein were diluted to 100 µl in TBS buffer (25 mM Tris, 150 mM NaCl, pH 7.2) and loaded into a 96 well plate, after which 100 µl of working solution was added to each well. Fluorescence activities (excitation 485 nm/emission 538 nm) were measured using a Bio-Tek Synergy HT (Winooski, VT) with readings taken every 5 minutes for 1 hour at 37°C. tRNA binding assays and reverse transcriptase sequencing of rRNAs. Binding of deacylated [32P]tRNA Phe was used to assay the general capacity of polyU programmed ribosomes for tRNAs, while binding of [14C]Phe-tRNA Phe was used to assess the specific activities of purified polyU programmed ribosomes. Competition experiments examined the ability of polyU programmed ribosomes pre-saturated with deacylated tRNA Phe to bind Ac-[14C] Phe-tRNA Phe to the ribosomal P-site. Control experiments utilized polyU programmed ribosomes and Ac-[14C]Phe-tRNA Phe alone to monitor specific activity of tRNA binding to the P-site. tRNA/ribosome binding reactions were performed as previously described17 with minor modifications. To prepare [32P]tRNA Phe, deacylated yeast tRNA Phe (Sigma, St. Louis) was end labeled
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using a KinaseMax kit (Ambion, Austin, TX) in the presence of g[32P]ATP.18 Ribosome master mixes contained 350 µl of 2X binding buffer (80 mM Tris-HCl, pH 7.4, 160 mM NH4Cl, 15 mM Mg(CH3COOH)2, 6 mM β-mercaptoethanol), 35 µl of poly(U) (10 mg/ml), 350 pmol of ribosomes and water to 700 µl. Six pairs of reactions were set up containing 25, 50, 100, 200, 400 and 800 pmol of [32P]-tRNA Phe to which 50 µl of ribosome mixture was added. Reactions were incubated at 30°C for 20 minutes, loaded onto premoistened Millipore HA (0.45 µ) filters (Billerica, MA), and subjected to vacuum filtration. Each filter was washed twice with 3 ml of binding buffer and radioactivity was quantified using a scintillation counter. Binding of [14C]PhetRNA Phe to ribosomal A-sites, with P-sites filled with deacylated tRNA, was performed as previously described17 with the modification that an ammonium sulfate generated fraction containing mixture of yeast elongation factors was added to purified ribosomes as described.19 rRNAs extracted from purified ribosomes were directly sequenced using [32P] labeled primer 25-6 (5'-AAC References Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 2000; 289:905-20. 2. Wimberly BT, Brodersen DE, Clemons WM Jr, et al. Structure of the 30S ribosomal subunit. Nature 2000; 407:327-39. 3. Schluenzen F, Tocilj A, Zarivach R, et al. Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 2000; 102:615-23. 4. Yusupov MM, Yusupova GZ, Baucom A et al. Crystal Structure of the Ribosome at 5.5 A Resolution. Science 2001; 292:883-96. 5. Frank J. Single-particle reconstrution of biological macromolecules in electron microscopy—30 years. Quarterly Reviews of Biophysics 2009; (in press). 6. Dinman JD. The eukaryotic ribosome: current status and challenges. J Biol Chem 2009; 284:11761-5. 7. Palade GE, Siekevitz P. Liver microsomes; an integrated morphological and biochemical study. J Biophys Biochem Cytol 1956; 2:171-200. 8. Algire MA, Maag D, Savio P, et al. Development and characterization of a reconstituted yeast translation initiation system. RNA 2002; 8:382-97.
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CTG TCT CAC GAC GG-3') which binds from positions 2964 to 2959 of the 25S rRNA, and SuperscriptIII reverse transcriptase (Invitrogen, Carlsbad, CA). Reactions were resolved through an 8% urea-polyacrylamide denaturing gel. The region probed roughly begins at the 3' end of Helix 90, through Helices 92 and 91, back through the 5' side of Helix 90, and through the 3' half of Helix 89. Acknowledgements
We wish to thank all of the members of the Dinman laboratory, including Ashton Belew, Karen Jack, Sharmishtha Musalga, Sergey Sulima, Shivani Reddy and Michael Rhodin for their help and input on this project. This work was supported by grants to A.M. from the American Heart Association (AHA 0630163N) and to J.D.D. from the National Institutes of Health (5R01 GM058859-12). J.A.L. was supported by an American Reinvestment and Recovery Act of 2009 supplement to the parent grant (3R01GM058859-11S1).
Halbeisen RE, Scherrer T, Gerber AP. Affinity purification of ribosomes to access the translatome. Methods 2009; 48:306-10. LaRiviere FJ, Cole SE, Ferullo DJ, Moore MJ. A lateacting quality control process for mature eukaryotic rRNAs. Mol Cell 2006; 24:619-26. Maguire BA, Wondrack LM, Contillo LG, Xu Z. A novel chromatography system to isolate active ribosomes from pathogenic bacteria. RNA 2008; 14:18895. Rheinberger HJ, Sternbach H, Nierhaus KH. Three tRNA binding sites on Escherichia coli ribosomes. Proc Natl Acad Sci USA 1981; 78:5310-4. Triana F, Nierhaus KH, Chakraburtty K. Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites. Biochem Mol Biol Int 1994; 33:909-15. Oakes M, Aris JP, Brockenbrough JS, Wai H, Vu L, Nomura M. Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae. J Cell Biol 1998; 143:23-34. Rakauskaite R, Dinman JD. An arc of unpaired “hinge bases” facilitates information exchange among functional centers of the ribosome. Mol Cell Biol 2006; 26:8992-9002.
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16. Cole SE, LaRiviere FJ, Merrikh CN, Moore MJ. A convergence of rRNA and mRNA quality control pathways revealed by mechanistic analysis of nonfunctional rRNA decay. Mol Cell 2009; 34:440-50. 17. Meskauskas A, Petrov AN, Dinman JD. Identification of functionally important amino acids of ribosomal protein L3 by saturation mutagenesis. Mol Cell Biol 2005; 25:10863-74. 18. McGarry KG, Walker SE, Wang H, Fredrick K. Destabilization of the P site codon-anticodon helix results from movement of tRNA into the P/E hybrid state within the ribosome. Mol Cell 2005; 20:613-22. 19. Dresios J, Derkatch IL, Liebman SW, Synetos D. Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable. Biochemistry 2000; 39:7236-44.
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