Development of a Fluorescence Polarization Assay for Peptidyl–tRNA Hydrolase

Analytical Biochemistry 306, 8 –16 (2002) doi:10.1006/abio.2002.5700

Development of a Fluorescence Polarization Assay for Peptidyl–tRNA Hydrolase Paul D. Bonin and Laurence A. Erickson Pharmacia Corporation, Kalamazoo, Michigan 49001

Received October 1, 2001; published online June 5, 2002

Peptidyl–tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl–tRNAs that result from premature termination of translation. Historically, the hydrolyzing activity of Pth has been assayed with radiolabeled N-blocked aminoacyl–tRNAs in assay systems that require the separation of radiolabeled amino acid from the N-blocked aminoacyl–tRNA complex. In the present study, we describe the development of a kinetic fluorescence polarization (FP) assay that enables measurements of Pth activity without the need to separate bound and free tracer. The hydrolyzing activity of Pth was determined by measuring the change in polarization values that resulted from the cleavage of a fluorescently labeled substrate (BODIPYLys-tRNA Lys). The data were analyzed using an equation describing first-order dissociation and the results showed that the experimental data correlated well with the theoretical curve. A runs test of the residuals showed that the experimental data did not significantly differ from the first-order model. The assay is adaptable to a multiwell format and is sensitive enough to detect Pth-like activity in bacterial cell lysate. The Pth FP assay provides a homogeneous and kinetic format for measuring Pth activity in vitro. © 2002 Elsevier Science (USA)

The normal process of protein synthesis involves the formation of initiation complexes with mRNA, repeated cycles of protein elongation, and the release of the polypeptide chain by hydrolysis of peptidyl–tRNA when the ribosome encounters a termination signal on mRNA (1, 2). However, during the course of elongation of a protein, peptidyl–tRNAs can dissociate prematurely from the ribosome, resulting in polypeptide chain termination (3, 4). These sequestered tRNA molecules (peptidyl– tRNAs) are released in bacteria (5–7) and yeast (6, 8) by the esterase activity of peptidyl–tRNA hydrolase 8

(Pth), 1 and thermosensitive mutants have established that the ability of Pth to recycle tRNA from aborted peptidyl–tRNAs is essential in Escherichia coli (9, 10). In these temperature-sensitive mutants, it is thought that at the nonpermissive temperature, peptidyl– tRNAs accumulate in the cytoplasm and are toxic for the cell by either impairing the initiation of translation or slowing down protein synthesis by limiting the number of free tRNAs (1, 3, 4). Pth hydrolyzes peptidyl–tRNAs and N-blocked aminoacyl–tRNAs but does not hydrolyze unblocked aminoacyl–tRNAs (5, 6). In the past, the hydrolyzing activity of Pth has been assayed with radiolabeled N-blocked aminoacyl–tRNAs (a notable exception to this is formylmethionyl–tRNA, which is resistant to attack by Pth; 6, 11) and traditional assay systems have required the separation of released radiolabeled amino acid from the N-blocked aminoacyl–tRNA complex. This has been accomplished by a number of different separation techniques, including paper chromatography (5, 12), filtration (13–15), and centrifugation (16, 17). Obviously, assays that require separation techniques of this nature do not lend themselves readily to evaluating large numbers of samples. Fluorescence polarization (FP) is a unique analysis tool that provides a rapid, nonradioactive, homogeneous format for measuring equilibrium binding and enzyme catalysis (18 –21). FP involves the measurement of the amount of light emitted into a horizontal and vertical plane by a fluorescent molecule excited by a vertical plane of polarized light (18). The polarization value P is then calculated as the difference of the vertical and horizontal intensities divided by the sum of these intensities (18). In FP applications, the fluo1

Abbreviations used: Pth, peptidyl-tRNA hydrolase; FP, fluorescence polarization; Ni–NTA, nickel–nitrilotriacetic acid; Me 2SO, dimethyl sulfoxide; BSA, bovine serum albumin; DTT, dithiothreitol; TCA, trichloroacetic acid; Chaps, (3-[cholamidopropyl) dimethylammonio]-1-propane-sulfonate). 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

FLUORESCENCE ASSAY FOR PEPTIDYL-tRNA HYDROLASE

rescent label is generally attached to the smaller of the two interacting molecules to maximize changes of polarization when the labeled molecule is bound. Conversely, when a small molecule that is labeled with a fluorophore is cleaved from another molecule of equal or greater size a change in polarization will occur. We postulated that Pth-catalyzed hydrolysis of fluorophore-labeled lysine from N-blocked Lys-tRNA Lys would result in a measurable decrease in polarization. The major advantage of a FP assay for Pth is that it would not require the separation of bound and free tracer. In this report, we describe the development of a kinetic FP assay for measuring Pth activity in vitro. MATERIALS AND METHODS

Preparation of Staphylococcus aureus Pth and S. aureus lysyl–tRNA synthetase. Pth and lysyl–tRNA synthetase were purified from cell pellets of E. coli expressing either S. aureus His-tagged Pth or Histagged lysyl–tRNA synthetase by Ni–NTA affinity as previously described (22). Pth and lysyl–tRNA synthetase prepared in this manner were biologically active and greater than 95% pure. The specific activity of the lysyl–tRNA synthetase was 252,000 units/mg where 1 unit of aminoacyl–tRNA synthetase activity is defined as the amount that will activate and attach 1 pmol of labeled amino acid to tRNA in 10 min at 37°C. Preparation of E. coli lysate. E. coli (K12 wild-type HN817 from Hiroshi Nikaido) were inoculated into 5 ml of Lennox L broth and incubated at 35°C with shaking. Fermentation was carried out until turbidity at 600 nm reached approximately 3.5. At that time, the cells were harvested by centrifugation and the resulting cell pellet was resuspended in 0.5 ml of buffer containing 50 mM NaH 2PO 4 (pH 8.0), 300 mM NaCl, 10 mM ␤-mercaptoethanol, and 10% glycerol. The cells were then sonicated with a Virsonic 50 (Virtis Co., Gardiner, NY) set at 60% of maximum output by six sets of 10-s bursts of sonication followed by 10 s of cooling on ice. The ruptured cells were then centrifuged at 10,000g for 30 min at 4°C and total protein of the resulting supernatant was determined using Coomassie Plus protein assay reagent (Pierce, Rockford, IL). Preparation of acetylfluorescein-[3H]Lys-tRNALys. The method used to acylate and acetylate the substrate is a modification of the procedure described by Schmitt et al. (23). Lys-tRNA synthetase (500 units) was added to 1 A 260 unit of E. coli tRNA Lys (Sigma, St. Louis, MO) in assay buffer containing 20 mM Tris (pH 7.5), 10 mM magnesium chloride, 0.1 mM EDTA, 1 mM ATP, and a 0.1 mM mixture of unlabeled lysine and L-[4,5- 3H] lysine monohydrochloride (Amersham, Arlington Heights, IL; final sp act of 2000 cpm/pmol lysine) in a final reaction volume of 200 ␮l. Incubation was carried out at 37°C. After 30 min, the reaction was quenched

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by the addition of 20 ␮l of 3 M sodium acetate (pH 5.5) and precipitation with ethanol. Fluorescein labeling was accomplished by dissolving the tRNA pellet in 100 ␮l of ice-cold buffer containing 0.1 M sodium bicarbonate (pH 8.5), 60 ␮M fluorescein succinimidyl ester (PanVera, Madison, WI) and 10% Me 2SO followed by incubation on ice. After 30 min, the reaction was quenched by the addition of 20 ␮l of 3 M sodium acetate, pH 5.5, and precipitation with ethanol. The resulting pellet was dissolved in 80 ␮l of 5 mM sodium acetate (pH 5.5) and acetylation was achieved by adding 100 ␮l of dimethyl sulfoxide, 20 ␮l of glacial acetic acid, and 40 ␮l of acetic anhydride and incubating at 0°C for 45 min (substrate that was not acetylated skipped this step and went directly to the deaminoacylation step). After ethanol precipitation and centrifugation, the resulting tRNA pellet was dissolved in 200 ␮l of 10 mM copper sulfate and 0.36 M sodium acetate (pH 5.5) and incubated at 37°C (this procedure deaminoacylates nonacetylated aminoacyl–tRNAs). After 30 min, the sample was ethanol-precipitated and centrifuged. To remove remaining copper ions, the tRNA pellet was dissolved in 200 ␮l of buffer containing 20 mM sodium acetate (pH 5.5), 100 mM KCl, and approximately 50 ␮l of chelating resin (Chelex 100; Sigma). The mixture was incubated at room temperature with occasional shaking. After 15 min the sample was centrifuged at ⬃600g and the supernatant fluid was removed and saved. The Chelex beads were washed twice more with 100 ␮l of 20 mM sodium acetate (pH 5.5) and 100 mM KCl by centrifugation. The combined supernatant fluids were precipitated with ethanol and then stored at ⫺70°C. The specific activity of the substrate was 2000 cpm/pmol of acetylfluorescein-[ 3H]LystRNA Lys. Preparation of BODIPY-labeled Lys-tRNALys substrate. Preparation of the Bodipy-labeled Lys-tRNA Lys was performed as described above with the exceptions that (1) only unlabeled lysine (0.1 mM) was used to acylate the tRNA Lys and (2) the fluorophore labeling was performed with 100 ␮M BODIPY succinimidyl ester (Molecular Probes, Eugene, OR). Standard assay for peptidyl–tRNA hydrolase. The activity of Pth was assayed using acetylfluorescein[ 3H]Lys-tRNA Lys as substrate. Pth activity was measured at room temperature in 100-␮l assays containing 20 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM EDTA, the indicated amount of acetylfluorescein-[3H]Lys-tRNA Lys, 200 ␮g/ml BSA, 10 mM freshly prepared DTT, and the indicated concentration of Pth. At the indicated time, the reaction was quenched by the addition of 5% TCA and 80 ␮g of carrier RNA (yeast tRNA; Sigma). The samples were then centrifuged and the released acetylfluorescein[H 3]lysine was measured in the supernatant by scintillation counting. The data were analyzed using GraphPad

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PRISM (GraphPad Software, Inc., San Diego, CA) with the first-order association equation Y ⫽ Y max ⫻ (1-exp (⫺K ⫻ X)). Fluorescence polarization assay for peptidyl–tRNA hydrolase. Pth activity was measured at room temperature in 100-␮l assays containing 2 mM NaH 2PO 4 (pH 7.2), 10 mM MgCl 2, 10 mM DTT, 100 ␮g/ml bovine ␥-globulin, 10% glycerol, 2 mM Chaps, and the indicated amounts of fluorophore-labeled lysine–tRNA Lys and Pth. The reaction was monitored using a Beacon 2000 FP system (PanVera, Madison, WI) or a Polarion multiwell fluorescence polarization plate reader (Tecan, Research Triangle Park, NC) (Ex 490, Em 535). For experiments with the Polarion, the gain was set to 90 and the number of flashes was set to 15. Both Beacon and Polarion data were analyzed using GraphPad PRISM (GraphPad Software, Inc.) with the firstorder disassociation equation Y ⫽ Y max ⫻ exp (⫺K ⫻ X) ⫹ plateau. HPLC analysis of fluorophore-labeled substrates. For HPLC analysis, BODIPY-labeled lysine-tRNA Lys was hydrolyzed as described above for the standard assay for Pth. FluoroTect Green Lys (Lys-tRNA Lys labeled with BODIPY FL only at the ⑀ position; Promega, Madison, WI) was hydrolyzed by incubation in 0.1 M Tris (pH 8.0) for 30 min at 37°C. Reaction products were analyzed by HPLC using a slightly modified version of the method described by Cayama et al. (24). Samples were analyzed by HPLC with a Vydac C4 column (4.6 ⫻ 250 mm, particle size 5 ␮m) equilibrated in 20 mM ammonium acetate (pH 5.0) and 400 mM NaCl. The column was eluted with a 0 to 60% methanol gradient (prepared in the equilibration buffer) over 60 min at a flow rate of 0.5 ml/min. Fluorescence was monitored using a Waters 474 scanning fluorescence detector (Waters, Milford, MA) with excitation and emission wavelengths set at 490 and 515 nm, respectively. RESULTS

For our initial experiments, tRNA Lys was acylated with [ 3H]lysine and then labeled with fluorescein succinimidyl ester. The presence of the tritium label allowed us to measure Pth-catalyzed release of acetylfluorescein-[3H]Lys from acetylfluorescein-[3H]LystRNA Lys by following either the tritium (scintillation counting in the standard assay) or the fluorescein (FP assay) labels. In addition, the presence of the tritium label provided a means for quantifying the amount of lysine accepted by the tRNA Lys. Many different combinations of pH, reaction temperature, and reaction time were tested before the present fluorescent-labeling conditions were adopted. Using the labeling scheme described under Preparation of acetylfluorescein-[3H]LystRNA Lys, 50 – 60% of the [ 3H]Lys-tRNA Lys complex was

FIG. 1. Ability of Pth to hydrolyze acetylfluorescein-[ 3H]LystRNA Lys. Assays were initiated by the addition of 1.0 nM Pth to 10 nM acetylfluorescein-[ 3H]Lys-tRNA Lys and Pth activity was determined by either the standard or the FP assay. (A) Standard assay. After 10 min, the reaction was quenched by the addition of 5% TCA and carrier RNA and hydrolysis of the substrate was determined by scintillation counting and the data were analyzed using a first-order association equation as described under Materials and Methods. Experimental data (F), theoretical curve (—). (B) FP assay. Hydrolysis of the substrate in the absence (■) or presence (F) of 1.0 nM Pth was measured in real time by monitoring changes in fluorescence polarization using a Beacon 2000 FP system. For these experiments, the ␨ factor on the Beacon system was set to 0.870. The data were analyzed using the first-order disassociation equation described under Materials and Methods. Experimental data (F,■), theoretical curve (—).

labeled with fluorophore during the course of the reaction with an overall recovery of approximately 30% following the final acetylation step. As seen in Fig. 1A, fluorescein labeling of acetyl-[3H]Lys-tRNA Lys did not affect the ability of Pth to hydrolyze that substrate. In this experiment, Pth-catalyzed hydrolysis of 10 nM acetylfluorescein[ 3H]Lys-tRNA Lys was determined after separation of the released acetylfluorescein-[ 3H]lysine from the acetylfluorescein-[ 3H]Lys-tRNA Lys complex by centrifugation (described under Materials and Methods). The results demonstrate that Pth produced a time-dependent increase in the release of acetylfluorescein-[3H]lysine from the acetylfluorescein-[ 3H]Lys-tRNA Lys complex. The results were analyzed with a first-order association equation and showed that the experimental data correlated well with the theoretical curve (R 2 ⫽ 0.981; calculated rate constant 0.431 ⫾ 0.05 min ⫺1). Additionally, analysis of residuals with a runs test demonstrated that the experimental

FLUORESCENCE ASSAY FOR PEPTIDYL-tRNA HYDROLASE

FIG. 2. Effect of Pth on acetylated and nonacetylated fluoresceinlabeled [ 3H]Lys-tRNA Lys. Hydrolysis of 10 nM fluorescein-[ 3H]LystRNA Lys (i.e., no acetylation) was measured in the absence (F) or presence (Œ) of 1.0 nM Pth. Similarly, hydrolysis of 10 nM acetylfluorescein-[ 3H]Lys-tRNA Lys also was measured in the absence (E) or presence (}) of 1.0 nM Pth. In both cases, FP was determined using the Beacon 2000 FP system.

data did not significantly differ from the first-order association model used for the analysis (P ⫽ 0.508). The ability of Pth to hydrolyze fluorescein-labeled substrate also could be successfully monitored by measuring changes in polarization. As seen in Fig. 1B, 10 nM substrate acetylfluorescein-[3H]Lys-tRNA Lys exhibited a polarization value of 53 mP that did not significantly change during the course of the experiment. However, with the addition of Pth, a time-dependent decrease in polarization to approximately 43 mP was observed. Importantly, this change in polarization was determined without having to separate the freed acetylfluorescein[ 3H]lysine from the acetylfluorescein-[ 3H]Lys-tRNA Lys complex. In this case, the results were analyzed with a first-order disassociation equation and showed that experimental data correlated well with the theoretical curve (R 2 ⫽ 0.997; calculated rate constant 0.330 ⫾ 0.01 min ⫺1) and analysis of residuals with a runs test again demonstrated that the experimental data did not significantly differ from the model used to analyze the data (P ⫽ 0.373). The decrease in polarization observed during the course of this experiment was not associated with any detectable changes in total fluorescence intensity. As already stated, Pth hydrolyzes peptidyl–tRNAs and N-blocked aminoacyl–tRNAs but does not hydrolyze unblocked aminoacyl–tRNAs (5, 6). We confirmed this when we observed that unblocked fluorescein-labeled LystRNALys did not act as a substrate for Pth. The results presented in Fig. 2 show that a measurable change in polarization was detected only when Pth was added to acetylfluorescein-[3H]Lys-tRNA Lys. The addition of Pth to unblocked (no acetylation step during substrate preparation) fluorescein-labeled [3H]Lys-tRNA Lys resulted in no change in polarization during the course of the experiment. Thus, fluorescein labeling of Lys-tRNALys must be followed by N-blocking for the complex to act as a substrate for Pth.

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In addition to fluorescein, we also labeled LystRNA Lys with BODIPY succinimidyl ester. As with the fluorescein label, BODIPY labeling of Lys-tRNA Lys did not affect the ability of Pth to hydrolyze the substrate. In fact, labeling Lys-tRNA Lys with BODIPY succinimidyl ester resulted in a higher starting polarization value (170 mP) than when the substrate was labeled with fluorescein (53 mP). The results presented in Fig. 3A show that Pth catalyzed the hydrolysis of BODIPYlabeled Lys-tRNA Lys in a concentration- and time-dependent manner resulting in a seven- to ninefold decrease in polarization over the course of the experiment (as with fluorescein-labeled substrate, this change in polarization was not associated with changes in total fluorescence intensity). Analysis of residuals with a runs tests again demonstrated that the experimental data did not significantly differ from the model used to analyze the data (P ⱖ 0.289 for all curves). Since this is a kinetic assay, rate constants for various test samples (in this case purified Pth) are easily obtained by fitting the curves to an equation describing first-order disassociation. Accordingly, the rate constants ob-

FIG. 3. Ability of Pth to hydrolyze BODIPY-labeled Lys-tRNA Lys. (A) Assays were initiated by the addition of the indicated concentration of Pth to 10 nM BODIPY-labeled Lys-tRNA Lys and hydrolysis of the substrate was determined by FP using a Polarion multiwell fluorescence polarization plate reader and the data were analyzed using the first-order disassociation equation described under Materials and Methods. Experimental data (symbol), theoretical curve (—). (B) Rate constants calculated from the above data are plotted versus Pth concentration and analyzed by linear regression using GraphPad PRISM (GraphPad Software, Inc.). Experimental data (F), theoretical regression line (—).

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FIG. 4. Ability of Pth to hydrolyze acetylated and nonacetylated BODIPY-labeled Lys-tRNA Lys. Assays were initiated by the addition of 1.0 nM Pth to 10 nM either nonacetylated BODIPY-labeled LystRNA Lys (F) or acetylated BODIPY-labeled Lys-tRNA Lys (Œ). Hydrolysis of the substrates was measured by FP using a Polarion multiwell fluorescence polarization plate reader and the data were analyzed using the first-order disassociation equation described under Materials and Methods. Experimental data (F,Œ), theoretical curve (—).

tained for the data presented in Fig. 3A were plotted versus Pth concentration and, as seen in Fig. 3B, a strong linear correlation (R 2 ⫽ 0.999) between Pth activity and Pth concentration was observed for the concentrations of Pth used in this experiment (0.125 to 8 nM Pth). Surprisingly, unlike with fluorescein labeling, we observed that acetylation of Lys-tRNA Lys after it was labeled with BODIPY was not required for the complex to act as a substrate for Pth. The results presented in Fig. 4 show that Pth produced a similar decrease in polarization when added to BODIPY-labeled Lys-tRNA Lys regardless of whether the BODIPYlabeled Lys-tRNA Lys had been subjected to acetylation following fluorophore labeling. The calculated rate constants for Pth-catalyzed hydrolysis of BODIPY-labeled Lys-tRNA Lys prepared with (0.198 ⫾ 0.002 min ⫺1) or without (0.148 ⫾ 0.001 min ⫺1) acetylation were similar. This result suggests that under the labeling conditions presented here, BODIPY succinimidyl ester is not interacting only with the ⑀-amine of the lysine of Lys-tRNA Lys but also with its ␣-amine. Therefore, it was possible that two species of the BODIPY-labeled Lys-tRNA Lys substrate were present in the above reactions: one species that is labeled with BODIPY only on the ␣-amine of the Lys-tRNA Lys and one that is labeled with BODIPY on both the ⑀- and the ␣-amines of the lysine. To investigate this possibility, we examined the products of Pth-catalyzed hydrolysis of the BODIPYlabeled Lys-tRNA Lys by HPLC. As seen in Fig. 5A, BODIPY-labeled Lys-tRNA Lys elutes as a single major peak with a retention time of 33.7 min. A small peak (⬍5% of the total fluorescence in the sample) eluting at 50.8 min (coincident with the elution time of free BODIPY) also was observed. As expected, treatment of the substrate with Pth produced complete hydrolysis of the BODIPY-labeled Lys-tRNA Lys and resulted in the

formation of a single product peak (representing 97% of total fluorescence in the sample) with an elution time of 43.4 min. For comparison, we also examined the hydrolysis of a commercially prepared BODIPY-labeled Lys-tRNA Lys that is labeled only at the ⑀ position of lysine (FluoroTect Green Lys). Because the ␣-amine of the lysine of FluoroTect Green Lys is not blocked, this molecule does not act as a substrate for Pth. However, hydrolysis of the molecule can be achieved by incubation at 37°C under basic pH conditions. As seen in Fig. 5B, FluoroTect Green Lys elutes as a single major peak with a retention time of 36.7 min. Treatment with basic pH at 37°C resulted in complete deacylation of the molecule and in the formation of a single product peak (representing 96% of the total fluorescence in the sample) with an elution time of 48.1 min. The K m of BODIPY-labeled Lys-tRNA Lys for Pth was determined from initial rate measurements that were made during five separate experiments and an example of the data is shown in Fig. 6. Data gathered during the first 75 s of the experiment shown in Fig. 6A were analyzed by linear regression and, as shown in Fig. 6B, there was a strong correlation between the experimental data and the theoretical line (R 2 ⱖ 0.95). Initial

FIG. 5. HPLC analysis of BODIPY-labeled Lys-tRNA Lys. (A) The reaction was initiated by the addition of 2.0 nM Pth to 10 nM BODIPY-labeled Lys-tRNA Lys. After 30 min at 24°C, the reaction products were analyzed by HPLC as described under Materials and Methods. BODIPY-labeled Lys-tRNA Lys (—), Pth-treated BODIPYlabeled Lys-tRNA Lys (- - -). (B) The reaction was initiated by incubating 10 nM FluoroTect Green Lys in 0.1 M Tris (pH 8.0) at 37°C. After 30 min, the reaction products were analyzed by HPLC as described under Materials and Methods. FluoroTect Green Lys (—), FluoroTect Green Lys treated with basic pH (- - -).

FLUORESCENCE ASSAY FOR PEPTIDYL-tRNA HYDROLASE

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labeled Lys-tRNA Lys determined in this manner was found to be 127 ⫾ 2.6 nM (n ⫽ 5). The sensitivity and utility of the Pth FP assay was demonstrated by the ability of the assay to detect Pthlike activity in bacterial cell lysate. The results presented in Fig. 7A show that bacterial lysate from E. coli catalyzed the hydrolysis of BODIPY-labeled LystRNA Lys in a concentration- and time-dependent manner (by 2 h, Pth-like activity could be detected in as little as 1.5 ␮g/ml of cell lysate). The curves were analyzed with a first-order model of decay and analysis of the residuals with a runs test demonstrated that as for purified Pth, the experimental data did not significantly differ from the first-order model used to analyze the data (P ⱖ 0.357 for all curves). The rate constants obtained for the data presented in Fig. 7A were plotted versus lysate concentration and, as seen in Fig. 7B, a strong linear correlation (R 2 ⫽ 0.998) between hydrolyzing activity and bacterial lysate concentration was observed for the concentrations of lysate used in this experiment (1.56 to 100 ␮g/ml). As with purified Pth

FIG. 6. K m determination of BODIPY-labeled Lys-tRNA Lys for Pth. (A) Assays were initiated by the addition of 1.0 nM Pth to 50 (F), 100 (E), 200 (Œ), 400 (‚), 800 (■), and 1600 nM (䊐) BODIPY-labeled Lys-tRNA Lys and hydrolysis of the substrate was determined by FP using a Polarion multiwell fluorescence polarization plate reader as described under Materials and Methods. (B) Data gathered during the first 75 s of the experiment shown in A were analyzed by linear regression using GraphPad PRISM (GraphPad Software, Inc.). Experimental data (symbols), theoretical regression lines (—). (C) Initial rates determined from the slope of the lines presented in B were plotted versus substrate concentration and K m was determined by analyzing the data with the equation Y ⫽ a ⫻ X/b ⫹ X (where a is V max and b is K m) using TableCurve 2D (Jandel Scientific, San Rafael, CA). Experimental data (F), theoretical curve (—).

rates of Pth-catalyzed hydrolysis of BODIPY-labeled Lys-tRNA Lys were determined from the slopes of these lines, plotted versus substrate concentration, and then analyzed with iterative nonlinear least-squares fits of the Michaelis–Menten equation. The results are presented in Fig. 6C and show that a strong correlation between the experimental data and the theoretical curve was observed (R 2 ⫽ 0.998). The K m of BODIPY-

FIG. 7. Determination of Pth-like activity in bacterial cell lysate. (A) Assays were initiated by the addition of the indicated concentrations of E. coli lysate to 10 nM BODIPY-labeled Lys-tRNA Lys and hydrolysis of the substrate was determined by FP using a Polarion multiwell fluorescence polarization plate reader and the data were analyzed using the first-order disassociation equation described under Materials and Methods. Experimental data (symbols), theoretical curve (—). (B) Rate constants calculated from the above data are plotted versus lysate concentration and analyzed by linear regression using GraphPad PRISM (GraphPad Software, Inc.). Experimental data (F), theoretical regression line (—).

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FIG. 8. Effect of magnesium chloride on the hydrolyzing activity of E. coli lysate. Assays were initiated by the addition of 5.0 ␮g/ml of E. coli lysate to 10 nM BODIPY-labeled Lys-tRNA Lys either in the presence (Œ) or in the absence (F) of 10 mM MgCl 2 in the assay buffer. Hydrolysis of the substrate was determined by FP using a Polarion multiwell fluorescence polarization plate reader as described under Materials and Methods.

(22), the Pth-like activity detected in E. coli cell lysate was dependent on the presence of magnesium chloride. In the absence of magnesium chloride, lysate prepared from E. coli did not produce any measurable hydrolysis of BODIPY-labeled Lys-tRNA Lys (as measured by changes in total polarization; Fig. 8). DISCUSSION

In the present report, we describe the development of a fluorescence polarization assay to measure the activity of peptidyl–tRNA hydrolase. This assay provides an important improvement over previous assays for Pth in that it does not require the separation of bound and free tracer. Additionally, by using a fluorescent label to monitor Pth activity, we have eliminated the need to deal with regulatory issues associated with the use of radioactive material. In FP applications, the fluorescent label is generally attached to the smaller of the two interacting molecules to maximize changes of polarization when the labeled molecule is bound (18 –20). The development of the Pth FP assay hinged on our ability to label the amino acid moiety of the aminoacyl–tRNA complex with a fluorescent molecule. Lys-tRNA Lys was chosen since lysine’s aliphatic ⑀-amine group is moderately basic and reacts with most acylating agents. Although a host of fluorescent probes that can be attached through covalent or noncovalent interactions are commercially available, fluorescent succinimidyl esters were chosen because they are good reagents for amine modification and because the amide bonds they form are as stable as peptide bonds (25). Additionally, a previous study had demonstrated that the ⑀-amine of lysine could be efficiently labeled with fluorescein using succinimidyl ester chemistry (26). Unfortunately, at the optimum pH (8.5–9.0) for amine modification by succinimidyl esters, the Lys-tRNA Lys complex becomes

unstable (27). Aminoacyl–tRNAs have been shown to be rapidly hydrolyzed under basic conditions and, therefore, it was necessary to develop a strategy that produced low deacylation rates of Lys-tRNA Lys while retaining acceptable rates of fluorescein labeling. This was accomplished by lowering the temperature of the labeling reaction to 4°C and by allowing the reaction to progress for no longer than 30 min at pH 8.5. Using those conditions, an acceptable level of fluorescent labeling of the [ 3H]Lys-tRNA Lys complex was achieved. After the final acetylation step, the substrate (acetylfluorescein-[ 3H]Lys-tRNA Lys) became remarkably stable and could be frozen and thawed several times without significant deacylation of the complex (this also was true of the BODIPY-labeled substrate). Initially, we postulated that Pth-catalyzed hydrolysis of a fluorophore-labeled N-blocked Lys-tRNALys would result in a measurable decrease in polarization. This was indeed the case as Pth-catalyzed hydrolysis of fluorescein-labeled acetyl-[3H]Lys-tRNA Lys consistently produced a measurable, although modest, decrease in fluorescence polarization. Importantly, it could be demonstrated that data from Pth-catalyzed hydrolysis of fluorescein-labeled acetyl-[ 3H]Lys-tRNA Lys exhibited first-order kinetics and resulted in similar rate constants whether monitored by the tritium (scintillation counting) or the fluorescein (FP) label. Changing the label to BODIPY succinimidyl ester resulted in an assay that produced similar first-order kinetics while providing an impressive improvement in the change in polarization in the assay. Unexpectedly, it also alleviated the need to acetylate the substrate following fluorophore labeling. This is in contrast to fluorescein-labeling of Lys-tRNALys, which must be followed by N-blocking (acetylation) for the labeled Lys-tRNALys to act as a substrate for Pth. Because Pth does not hydrolyze unblocked aminoacyl–tRNAs (5, 6), we must infer that during labeling of Lys-tRNALys, BODIPY succinimidyl ester interacts with not only the ⑀-amine of lysine but also its ␣-amine. This is supported by the fact that Pth could hydrolyze the BODIPY-labeled Lys-tRNALys substrate even when the acetylation step was not performed. Unfortunately, this finding suggested that two species of BODIPY-labeled Lys-tRNALys substrate might be present in the FP reactions: one species that was labeled with BODIPY on the ␣-amine of the Lys-tRNALys and another that was labeled with BODIPY on both the ⑀- and the ␣-amines of the lysine. To investigate this issue, the product(s) of Pth-catalyzed hydrolysis of the BODIPY-labeled Lys-tRNA Lys substrate was analyzed by HPLC. The results showed that Pth catalyzed the complete hydrolysis of the substrate and that only one fluorescently labeled product peak was produced (i.e., only one species of BODIPY-labeled Lys-tRNALys substrate was present in the reaction). Based on retention times, the product of this reaction was shown to be larger than the product (lysine labeled with one BODIPY molecule) of the deacy-

FLUORESCENCE ASSAY FOR PEPTIDYL-tRNA HYDROLASE

lation reaction of FluoroTect GreenLys. Taken together, these results demonstrate that (1) only one species of BODIPY-labeled Lys-tRNALys substrate is present in the experiments described in this report and (2) the BODIPY-labeled Lys-tRNALys substrate is almost certainly labeled with BODIPY on both the ⑀- and the ␣-amines of lysine. The fact that BODIPY succinimidyl ester interacts with the ␣-amine of lysine could also explain the improvement in the change in polarization seen when Pth hydrolyzes BODIPY-labeled Lys-tRNA Lys compared to its hydrolysis of fluorescein-labeled Lys-tRNA Lys. The most important aspect of developing a fluorescence polarization assay is the development of a suitable fluorescent tracer which gives a maximum polarization change and minimum “propeller effect” (29). Propeller effect is a term used to describe the phenomenon whereby, although binding has occurred, little polarization shift is observed. This is thought to be the result of an uncoupling of the fluorophore and the binding site due to a long flexible linkage (29). In the present case, the molecules that are bound together can be thought of as the fluorescently labeled amino acid that is attached to the tRNA molecule. Therefore, it seems likely that the labeling of Lys-tRNA Lys with fluorescein is an example of propeller effect since in this case, the fluorescein label on the ⑀-amine of lysine is isolated from the larger tRNA molecule by the intervening four carbons of the lysine molecule, thus creating a flexible linkage from which the fluorescein molecule can “propel.” The consequence of this is that the starting polarization value of the fluorescein-labeled Lys-tRNA Lys is low. In contrast, the interaction of BODIPY with the ␣-amine as well as the ⑀-amine of lysine in Lys-tRNA Lys puts the fluorescent label in closer proximity to the tRNA molecule. The resulting shorter linkage between the BODIPY label and the tRNA molecule likely reduces the apparent propeller effect seen when the fluorescein label is used, thus resulting in a higher starting polarization value for the BODIPY-labeled Lys-tRNA Lys. Consequently, a larger change in polarization is then observed when the substrate is hydrolyzed by Pth. Surprisingly, labeling LystRNA Lys with BODIPY succinimidyl ester also resulted in an improvement in the affinity of Pth for the substrate, with the K m of BODIPY-labeled Lys-tRNA Lys at least 10-fold lower than the reported K m of other Nblocked aminoacyl substrates (6, 11, 27, 28). The Pth FP assay was amenable to a multiwell format and since the assay is of a kinetic design, rate constants for numerous samples can be easily determined and readily compared. Whether the activity of purified Pth or bacterial cell lysate was analyzed, a strong linear correlation existed between observed changes in FP (hydrolysis of the BODIPY-labeled LystRNA Lys) and the amount of material added to the

15

reaction. The ability of the Pth FP assay to enable accurate measurement of slight differences in Pth-like activity is an advantage particularly when examining bacterial cell lysates for Pth-like activity. Some investigators have been forced to seek indirect means of demonstrating Pth-like activity (or lack thereof) in some types of bacterial cell lysates (30, 31). In this regard, the ability of the Pth FP assay to measure Pth-like activity in as little as 1.5 ␮g/ml of bacterial cell lysates could be of particular use. Of course without further characterization, it is not possible to attribute the hydrolyzing activity measured in bacterial lysate to Pth. One expected characteristic of Pth in E. coli is that the hydrolyzing activity of the enzyme would be magnesium-dependent (23). As with purified Pth, the activity contained in the lysate of E. coli measured in this study was shown to be dependent on the presence of magnesium, thus suggesting that as expected, this hydrolyzing activity was indeed due to the presence of Pth in the lysate. In conclusion, the homogeneous, kinetic format of the Pth FP assay provides a relatively easy and accurate means by which large numbers of samples can be assayed for Pth-like activity in vitro. ACKNOWLEDGMENTS The authors thank Dennis Epps, Paul Tomich (Discovery Technologies, Pharmacia, Kalamazoo, MI), and Roger Poorman (Protein Science, Pharmacia, Kalamazoo, MI) for helpful discussions about fluorescence polarization.

REFERENCES 1. Menninger, J. R. (1976) Peptidyl-transfer RNA dissociates during protein synthesis from ribosomes of E. coli. J. Biol. Chem. 251, 3392–3398. 2. Menninger, J. R. (1979) Accumulation of peptidyl-tRNA is lethal to Escherichia coli. J. Bacteriol. 137, 694 – 696. 3. Atherly, A. G. (1978) Peptidyl-transfer RNA hydrolase prevents inhibition of protein synthesis initiation. Nature 275, 769 –770. 4. Chapeville, F., Yot, P., and Paulin, D. (1969) Enzymatic hydrolysis of N-acylaminoacyl transfer RNAs. Cold Spring Harbor Symp. Quant. Biol. 34, 493– 498. 5. Cuzin, F., Kretchmer, N., Greenberg, R. E., Hurwitz, R., and Chapeville, F. (1967) Enzymatic hydrolysis of N-substituted aminoacyl-tRNA. Proc. Natl. Acad. Sci. USA 58, 2079 –2086. 6. Ko¨ ssel, H., and RajBhandary, U. L. (1968) Studies on polynucleotides. LXXXVI. Enzymatic hydrolysis of N-acylaminoacyltransfer RNA. J. Mol. Biol. 35, 539 –560. 7. Ko¨ ssel, H. (1970) Purification and properties of peptidyl-tRNA hydrolase from Escherichia coli. Biochim. Biophys. Acta 204, 191–202. 8. Jost, J. P., and Bock, R. M. (1969) Enzymatic hydrolysis of N-substituted aminoacyl transfer ribonucleic acid in yeast. J. Biol. Chem. 244, 5866 –5873. 9. Atherly, A. G., and Menninger, J. R. (1972) Mutant Escherichia coli strain with temperature sensitive peptidyl-transfer RNA hydrolase. Nature 240, 245–246. 10. Refugio Garcia-Villegas, M., De La Vega, F. M., Galindo, J. M., Segura, M., Buckingham, R. H., and Guarneros, G. (1991) Pep-

16

11.

12.

13.

14.

15. 16. 17.

18.

19.

20.

21.

BONIN AND ERICKSON tidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis. EMBO J. 10, 3459 –3555. Schulman, L., and Pelka, H. (1975) The structural basis for the resistance of Escherichia coli formylmethionyl transfer ribonucleic acid to cleavage by Escherichia coli peptidyl transfer ribonucleic acid hydrolase. J. Biol. Chem. 250, 542–547. De Groot, N., Groner, Y., and Lapidot, Y. (1969) Peptidyl-tRNA. VII. Substrate specificity of peptidyl-tRNA hydrolase. Biochim. Biophys. Acta 186, 286 –296. De Groot, N., Panet, A., and Lapidot, Y. (1968) Enzymatic hydrolysis of peptidyl-tRNA. Biochem. Biophys. Res. Commun. 31, 37– 42. Vogel, Z., Zamir, A., and Elson, D. (1968) On the specificity and stability of an enzyme that hydrolyzes N-substituted aminoacyltransfer RNA’s. Proc. Natl. Acad. Sci. USA 61, 701–707. Zucker, W. V. (1975) A new method for assaying peptidyl-tRNA hydrolase. Anal. Biochem. 63, 522–527. Shiloach, M., Lapidot, Y., and De Groot, N. (1975) The specificity of peptidyl-tRNA hydrolase from E. coli. FEBS Lett. 57, 130 –133. Dutka, S., Meinnel, T., Lazennec, C., Mechulam, Y., and Blanquet, S. (1993) Role of the 1–72 base pair in tRNAs for the activity of Escherichia coli peptidyl-tRNA hydrolase. Nucleic Acids Res. 21, 4025– 4030. Checovich, W. J., Bolger, R. E., and Burke, T. (1995) Fluorescence polarization—A new tool for cell and molecular biology. Nature 375, 254 –256. Lundblad, J. R., Laurance, M., and Goodman, R. H. (1996) Fluorescence polarization analysis of protein–DNA and protein– protein interactions. Mol. Endocrinol. 10, 607– 612. Burke, T., Bolger, R., Checovich, W., and Lowery, R. (1996) in Phage Display of Peptides and Proteins (Kay, B. K., Winter, J., and McCafferty, J., Eds.), pp. 305–326, Academic Press, San Diego. Levine, L. M., Michener, M. L., Toth, M. V., and Holwerda, B. C. (1997) Measurement of specific protease activity utilizing fluorescence polarization. Anal. Biochem. 247, 83– 88.

22. Bonin, P. D., Choi, G. H., Trepod, C. M., Mott, J. E., Lyle, S. B., Cialdella, J. I., Sarver, R. W., Marshall, V. P., and Erickson, L. A. (2001) Expression, purification, and characterization of peptidyltRNA hydrolase from Staphylococcus aureus. Protein Expression Purif. 24, 123–130. 23. Schmitt, E., Mechulam, Y., Fromant, M., Plateau, P., and Blanquet, S. (1997) Crystal structure at 1.2 Å resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase. EMBO J. 16, 4760 – 4769. 24. Cayama, E., Yepez, A., Rotondo, F., Bandeira, E., Ferras, A., and Triana-Alonso, F. J. (2000) New chromatographic and biochemical strategies for quick preparative isolation of tRNA. Nucleic Acids Res. 28, e64. 25. Haugland, R. P. (1996) in Handbook of Fluorescent Probes and Research Chemicals, 6th ed., Molecular Probes, Eugene, OR. 26. Banks, P. R., and Paquette, D. M. (1995) Comparison of three common amine reactive fluorescent probes used for conjugation to biomolecules by capillary zone electrophoresis. Bioconjugate Chem. 6, 447– 458. 27. Gillam, I., Blew, D., Warrington, R. C., von Tigerstrom, W., and Tener, G. M. (1968) General procedure for the isolation of specific transfer ribonucleic acids. Biochemistry 10, 3459 –3468. 28. Shiloach, J., Bauer, S., De Groot, N., and Lapidot, Y. (1975) The influence of the peptide chain length on the activity of peptidyltRNA hydrolase from E. coli. Nucleic Acids Res. 2, 1941–1950. 29. Nasir, M. S., and Jolley, M. E. (1999) Fluorescence polarization: An analytical tool for immunoassay and drug discovery. Comb. Chem. High Throughput Screen. 2, 177–190. 30. Heurgue´ -Hamard, V., Mora, L., Guareros, G., and Buckingham, R. H. (1996) The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA Lys. EMBO J. 15, 2826 –2833. 31. Jenkins, B. D., and Barkan, A. (2001) Recruitment of a peptidyltRNA hydrolase as a facilitator of group II intron splicing in chloroplasts. EMBO J. 20, 872– 879.