The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

Purification of ribosomes and S100 subunits.Ribosomes and S100 subunits were prepared essentially as described previously (1). Cultures of Escherichia coli MG1655 and its ΔybeY derivative were grown in LB broth (Difco) medium at 37°C to an optical density at 600 nm (OD₆₀₀) of 0.5 and immediately poured onto an equal volume of crushed ice. Pellets were washed once with 30 ml buffer consisting of 20 mM HEPES-KOH (pH 7.5), 30 mM NH₄Cl₂, 6 mM MgCl₂, and 4 mM β-mercaptoethanol and resuspended in 30 ml of the same buffer. Cells were disrupted using a French press, and the lysates were centrifuged for 1 h at 4°C at 32,000 × g using a fixed-angle 70Ti rotor to remove cell debris and membranes. The upper three-fourths of the supernatant was centrifuged for 17 h at 70,000 × g at 4°C to pellet ribosomes. The top three-fourths of the supernatant was centrifuged once more for 4 h at 150,000 × g at 4°C. The top three-fourths of this supernatant was dialyzed in a dialysis bag with a molecular weight cutoff of 3,500 against 100 volumes of the same buffer for 2 h with three buffer changes. The dialyzed S100 subunit was concentrated using a centrifugal filter unit with a molecular weight cutoff of 3,500 (Amicon Ultra) until a protein concentration of 3 to 5 μg/μl was reached.

Ribosomes were prepared from the 70,000 × g ultracentrifugation pellet by the following procedure. The pellet was washed twice with 1.5 ml buffer containing 20 mM HEPES-KOH (pH 7.5), 200 mM NH₄Cl₂, 1 mM MgCl₂, and 4 mM β-mercaptoethanol. Next, the pellet was scraped off with a plastic sliver and completely dissolved by gentle shaking in 1.5 ml of the same buffer for 2 h at 4°C.

In order to purify the ribosomal subunits, crude ribosome extract (the dissolved 70,000 × g pellet) with an A ₂₆₀ of 100 was loaded onto a 10 to 30% sucrose gradient prepared with the same buffer. The 30S and 50S subunits were separated by ultracentrifugation in an SW28 swinging-bucket rotor for 17 h at 48,000 × g at 4°C. Gradients were fractionated from top to bottom, and peaks corresponding to the 30S and 50S ribosomal subunits were separately collected to avoid any cross contamination between the two species. The 30S and 50S subunits were concentrated by ultracentrifugation in a 70Ti rotor for 20 h at 190,000 × g at 4°C, and the pellet was treated as described for the 70,000 × g pellet. Ribosomes were quantified by A ₂₆₀ measurements. In a typical preparation, a concentration of 0.05 to 0.2 U of A ₂₆₀/μl was achieved.

In vitro transcription-translation assays. In vitro transcription-translation reactions were in a final volume of 30 μl containing 60 mM HEPES-KOH (pH 7.5); 14 mM magnesium acetate; 200 mM potassium glutamate; 30 mM ammonium acetate; 2 mM dithiothreitol; 4% PEG 8000; 1.5 mM ATP; 1 mM CTP, GTP, and UTP; 0.7 mM cyclic AMP; 80 mM creatine phosphate; 0.125 mg/ml creatine kinase (Sigma); 0.18 mg/ml total E. coil tRNA (Sigma); and 1 mM each amino acid. One hundred nanograms of pIVEX (Roche) with a cloned luciferase gene, 10 U of T7 RNA polymerase (New England Biolabs), 0.33 A ₂₆₀ U of the 30S subunit, 0.66 A ₂₆₀ U of the 50S subunit, and 16.2 μg of the S100 subunit were then added to initiate the reactions. Incubation was at 37°C, reactions were stopped by the addition of 50 μl chloramphenicol at a final reaction mixture concentration of 0.5 mM, and the reaction mixture was transferred to 4°C to avoid unfolding of the translated luciferase. In order to measure luciferase activity, 10-μl samples were removed to a white opaque microplate, luciferin and ATP (Promega luciferase assay system) were added, and the light produced was measured using a VERITAS microplate luminometer (Turner Bio Systems).

Analysis of ribosome profiles.Ribosome profiles were analyzed as previously described (10, 19). Cultures of E. coli MG1655 with chromosomally FLAG×3-taggd ybeY or rpsT were grown exponentially at 37° to an OD₆₀₀ of 0.5. The cultures were cooled on ice for 15 min and centrifuged for 5 min at 5,000 × g at 4°C. Pellets were resuspended in a buffer containing 20 mM HEPES-KOH (pH 7.5), 200 mM NH₄Cl₂, 1 mM MgCl₂, and 4 mM β-mercaptoethanol. Gentle lysis was achieved by addition of lysozyme (0.75 mg/ml final) and incubation on ice for 3 min, followed by three freeze-thaw cycles with liquid N₂. The lysates were cleared by centrifugation for 10 min at 20,000 × g at 4°C. Thirteen A ₂₆₀ units was applied to 5 to 25% sucrose gradients. The lysates were separated on sucrose gradients prepared in a buffer consisting of 20 mM HEPES-KOH, 200 mM NH₄Cl₂, 1 mM MgCl₂, and 4 mM β-mercaptoethanol by centrifugation for 5 h at 187,000 × g at 4°C in an LE-70 ultracentrifuge (Beckman). Gradients were fractionated into 48 fractions from top to bottom and read in UV-transparent microplates (Costar), and A ₂₆₀ was read in a Spectra-Max 190 (Molecular Devices). Fractions were pooled and trichloroacetic acid (TCA) precipitated in order to detect YbeY, S20, and β-galactosidase (β-gal) as described below.

FLAG tagging for immunoblotting.Chromosomal epitope tagging was achieved using the system described by Uzzau et al. (20).

SDS-PAGE and immunoblotting.Gel electrophoresis was carried out according to published protocols using sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting was performed according to standard procedures to detect YbeY and ribosomal protein S20 tagged with the FLAG×3 epitope at the C terminus. Mouse monoclonal antibodies specific for luciferase, β-gal, and FLAG×3 (Sigma) were used as primary antibodies, and horseradish peroxidase-conjugated anti-mouse IgG (Sigma) was used as the secondary antibody. The EZ-ECL chemiluminescence detection kit (Biological Industries) was used for development, and the immunoblots were scanned using an ImageScanner (Amersham Pharmacia Biotech).

Reduced in vitro activity of the ΔybeY translation machinery. In vivo studies of the ΔybeY mutant (17) suggest that YbeY directly affects ribosomes or ribosome-associated factors that function in the translation machinery. This result could reflect a direct involvement of YbeY in the translation process. However, it is possible that YbeY affects the cellular translation machinery indirectly. For instance, depletion of nucleotides (7), starvation for an amino acid (15), and even a specific defect in cotranslational protein targeting of integral inner membrane proteins (6) can all lead to an apparent general translational defect.

In order to distinguish between the two alternatives, the translation machinery was prepared from wild-type and ΔybeY mutant cultures grown at 37°C and used for coupled in vitro transcription-translation assays as described in Materials and Methods. In these assays, translation efficiency was determined by measurements of the activity of the translated reporter protein—Photinus pyralis firefly luciferase. Figure 1 presents the results of luciferase activity measurements as a function of the reaction time of the translation machineries prepared from the wild type and the ΔybeY mutant. The results indicate that the activity of the wild-type system is 8- to 9-fold higher than that of the ybeY deletion mutant. This difference remains constant throughout the reaction, from 10 min to the end of the reaction after 90 min. The reaction reaches saturation after approximately 60 min in both the wild type and the ΔybeY mutant (Fig. 1).

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FIG. 1.

Reduced in vitro activity of the ΔybeY mutant translation machinery. The translation machinery was prepared from E. coli MG1655 wild-type cultures (filled circles) and from its ΔybeY mutant derivative (open circles) as described in Materials and Methods. Reaction mixtures were incubated at 37°C, and samples were collected at the indicated time points and kept on ice following the addition of a translation inhibitor. The activity of the translated luciferase was measured as described in Materials and Methods. The average and standard deviation of three independent experiments are presented for each time point.

The lower luciferase activity may indicate a lower luciferase protein level. However, it is also possible that this lower activity is due to severe misreading by the defective machinery, resulting in multiple translation errors, which lower the activity of the enzyme. To distinguish between these two possibilities, we determined the concentration of luciferase by Western blotting (Fig. 2). The data indicate that the concentration of luciferase is significantly reduced when it is translated from the ybeY machinery. These results also indicate that YbeY directly affects one of the components of the translation machinery.

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FIG. 2.

Immunoblot assay of luciferase produced by in vitro translation. The experiment was performed as described in the legend to Fig. 1. Luciferase levels were determined using SDS-PAGE separation, followed by Western blotting with mouse antiluciferase antibodies. Samples collected at the indicated time points from the experiment described in Fig. 1 were also used for luciferase protein level determinations.

YbeY is located in the S100 fraction.The translation machinery includes the ribosomes and the S100 fraction, which consists of all low-molecular-weight factors, such as translation factors (initiation, elongation, release, and recycling factors) and enzymes such as tRNA synthetases. In order to determine whether YbeY is in the S100 fraction or in the ribosomal fraction, we performed an analysis of ribosome profiles and detected the presence of YbeY in the various samples by immunoblotting. The addition of a FLAG epitope-encoding sequence to the endogenous copy of ybeY on the chromosome did not compromise the ability of this strain to grow at high temperature (data not shown). Therefore, it is reasonable to assume that FLAG-tagged YbeY behaves the same as wild-type YbeY in vivo. The results presented in Fig. 3 indicate that YbeY is found entirely in the soluble protein fractions, similar to β-galactosidase and in contrast to the ribosomal protein S20 (RpsT). Therefore, we can conclude that YbeY is not associated with the ribosomes in our preparations.

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FIG. 3.

YbeY does not comigrate with ribosomes. Cultures of E. coli MG1655 with chromosomally FLAG-tagged (20) ybeY and rpsT were grown in LB at 37°C to an OD₆₀₀ of 0.4 and then cooled down on ice for 15 min to allow runoff of nascent peptides. Next, lysates were prepared and 13 OD₂₆₀ units was separated through a 5 to 25% sucrose gradient by ultracentrifugation. Gradients were fractionated from top to bottom, and OD₂₆₀ was determined. The presence of YbeY, S20, and β-gal in the different fractions was determined by TCA concentration, SDS-PAGE separation, and immunoblotting as described in Materials and Methods.

The S100 fraction prepared from the ΔybeY mutant is as active as the S100 fraction prepared from the wild type.In order to determine which constituent of the translation machinery is impaired in the ΔybeY mutant, an in vitro translation assay was performed with all four possible combinations of ribosomes and S100 fractions. The results (Fig. 4) indicated that when wild-type ribosomes were supplemented with the S100 fraction prepared from the ΔybeY mutant, the luciferase activity was similar to that achieved when wild-type ribosomes were provided with the wild-type S100 fraction. This result implies that the S100 fraction prepared from the ΔybeY mutant is almost as active as the S100 fraction prepared from the wild type. The reciprocal experiment shows that low levels of luciferase activity are obtained wherever ΔybeY mutant ribosomes are present, irrespectively of the source of the S100 fraction. This result indicates that the ribosomes prepared from the ΔybeY mutant are defective.

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FIG. 4.

The S100 fraction prepared from the ΔybeY mutant is as active as the wild-type S100 fraction. In vitro translation reaction mixtures were incubated at 37°C for 60 min, and then the reactions were terminated as described in the legend to Fig. 1. Each tube contained one of the four different combinations of ribosomes and the S100 fraction prepared from the wild type or the ΔybeY mutant. The average and standard deviation of three independent experiments are presented for each reaction.

The lower activity of the ΔybeY mutant translation machinery stems from the 30S subunit.Further purification of the ribosomal fraction preparations of both the wild type and the ΔybeY mutant enables isolation of the 30S small ribosomal subunits and the 50S large ribosomal subunits and determination of which ribosomal subunit is defective in ybeY deletion mutants. An assay with the wild-type S100 subunit and all four possible combinations of the 30S and 50S subunits was performed, and the results are presented in Fig. 5 A. When wild-type the 30S fraction was supplemented with the ΔybeY mutant 50S subunit, the luciferase activity was approximately the same as the value achieved with the wild-type 50S subunit. However, addition of the ΔybeY mutant 30S subunit to the wild-type 50S subunit yielded a luciferase activity which was much lower, indicating that the 30S ribosomal subunit of the ΔybeY mutant is defective. This defect of the 30S ribosomal subunit could be shown with the S100 subunit from the ybeY deletion mutant as well (Fig. 5B).

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FIG. 5.

The translational defect of the ΔybeY mutant machinery stems specifically from the 30S fraction. In vitro translation reaction mixtures were incubated at 37°C for 60 min, and then the reactions were terminated as described in the legend to Fig. 1. Each tube contained the (A) wild-type or (B) ΔybeY mutant S100 fraction and one of the four different combinations of the 30S and 50S fractions prepared from the wild type and the ΔybeY mutant. The standard deviation of three independent experiments is presented for each reaction.