V O L . 8, N O . 2, F E B R U A R Y 1 9 6 9
Coggin, J. H., Loosemore, M., and Martin, W. R. (1966), J . Bacterioi. 92,446. Demain, A. L. (1964), Arch. Biochem. Biophys. 108,403. Dikstein, S., Bergmann, F., and Henis, Y. (1957), J. Biol. Chem. 224,67. Kalle, G. P., and Gots, J. S. (1964), Science 142,680. Kirby, K. S. (1956), Biochem. J. 64,405. Levin, G., Kalmus, A., and Bergmann, F. (1960), J. Org. Chem. 25,1752. Magasanik, B., and Karibian, D. (1960), J . Bioi. Chem.
235,2672. Mager, J., and Magasanik, B. (1960), J. Biol. Chem. 235,1474. Meybaum, W. (1939), Z. Physioi. Chem. 258,117. Roblin, R. O., Jr., Lampen, J. O., English, J. P., Cole, Q. P., and Vaughan, J. R., Jr. (1945), J. Am. Chem. Soc. 67,290. Roodyn, D. B., and Mandel, H. G. (1960), J. Biol. Chem. 235,2036. Traube, W. (1900), Chem. Ber. 33,3035.
Irreversible Thermal Denaturation of Escherichia coli Ribosomes" James W. Bodley
ABSTRACT: Irreversible thermal denaturation of high salt-washed Escherichia coli (B and Q13) ribosomes has been followed in terms of biological activity, sedimentation properties, and turbidity, as a function of solvent composition. In a buffer composed of 0.01 M Tris-0.01 M MgAcl (pH 7.4) ribosomes lose 50% of their ability to form polyphenylalanine from polyuridylic acid in 5 min at 57' (Td,5). In this temperature range 70s ribosomes are destroyed, presumably by aggregation. The sharpness of this thermal transition is illustrated by the fact that in 0.01 M Tris-0.01 M MgAcs several hours are required for 50% inactivation at 50°, while at 60" 50% inactivation occurs within 1 min. Reduction of the magM reduces the Td.5to nesium ion concentration to 53.5" and under these conditions the normal 50.5 and 30s subunits are converted into slower sedimenting particles ( ~ 3 5 - 4 0and 25s). Monovalent cations (K 3 NH4 > Na) in the presence of 0.01 M MgAcn (but not
T
he ribosomes of Escherichia coli are not only structurally complex but they have a complex role in protein synthesis. They are known to contain 3 RNA molecules and probably as many as 40 different protein molecules. Very little information, however, is available on the way in which these individual components interact to produce the structural and biological features of the ribosomal particle. Magnesium ion is essential to the maintenance of both the structural and functional integrity of the ribosome and several investigators have shown that ribosomes
* From the Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota 55455. Receiced August 19, 1968. This research was supported by grants from the University of Minnesota Graduate School and Cancer Coordinating Committee and from the American Cancer Society (P-455).
M MgAc2) protect ribosomes from thermal inactivation presumably by preventing aggregation. In 0.01 M Tris0.01 M MgAc2-0.1 M KCl (pH 7.4), ribosomes exhibit a Td,&of 60.5 O and the loss in biological activity is closely paralleled by the destruction of the 30s subunit. The effect of heating on ribosome activity is characterized by an initial increase in activity (variable with the preparation) followed by a rapid but decreasing rate of inactivation. No explanation is provided for this activation, but it apparently results from an increase in the number of active ribosomes and is not due to a relaxation of the ribosomal structure which would also lead to translational ambiguity. Evidence is presented which suggests that the observed denaturation does not result from enzymatic degradation. It is also shown that ribosomes can be lyophilized from a variety of buffers with little or no alteration of physical or functional properties.
undergo a characteristic and partially reversible unfolding upon the removal of magnesium ion (see Gavrilova et ai., 1966; Gesteland, 1966). Kaji et al. (1966) have shown that mild proteolysis of ribosomes progressively destroys their biological activity in the order, protein synthesis, aminoacyl-tRNA binding, mRNA binding, and ultimately causes irreversible dissociation into apparently normal 50s and 30s subunits without the release of appreciable soluble material. On the basis of chemical modification of the ribosome, Moore (1966) has concluded that amino groups of rRNA are also involved in the binding of mRNA and in the interaction of the subunits. Protein sulfhydryl groups have also been implicated in ribosomal structure and function (Wang and Matheson, 1966; Tamaoki and Miyazawa, 1967; Traut and Haenni, 1967). In addition to their role in binding mRNA and amino-
465
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acyl-tRNA it has recently been suggested that ribosomal protein is specifically responsible for peptide-bond formation (Monro, 1967). Recent experiments with reassembled, protein-deficient ribosomes (see Traub and Nomura, 1968) have also suggested unique functions for the various proteins. The objective of the present investigation was to examine the irreversible structural and functional changes accompanying thermal denaturation of E. coli ribosomes in the hope of gaining further insight into the form of the particle required for activity and the nature of the forces which maintain its structure and how they are effected by environment. The thermal denaturation of ribosomes has in the past received only limited attention (Saunders and Campbell, 1966; Tamaoki and Miyazawa, 1966; Miyazawa and Tamaoki, 1967) and no attempt has been made to correlate alterations in structural and functional parameters. Furthermore, cognizance should be taken of the activation of RNase I as a possible cause of thermal denaturation of ribosomes as suggested by Schell (1965). Recently, McLaughlin et al. (1968) have examined the thermal stability of mRNA-tRNA-ribosome complexes and shown that such complexes can undergo a thermally induced reversible dissociation and that among other factors the intrinsic stability of the ribosome under some conditions establishes an upper limit to the thermal stability of these complexes.
Materials and Methods
466
Materials. E. coli cell paste (B and Q13, late log) was purchased from General Biochemicals, Inc. IC-Labeled amino acids were obtained from New England Nuclear Corp. The polynucleotides were purchased from Miles Laboratories and ATP, GTP, and phosphoenolpyruvate were purchased from Sigma. Pyruvate kinase was purchased from C. F. Boehringer und Soehne. Preparation of Ribosomes and S-100. All operations were carried out between 0 and 4" unless otherwise specified and all pH measurements were made at ambient temperature, ca. 22". Cell paste was thawed and washed twice with a buffer containing 0.01 M Tris-Cl-O.01 M MgAc2(pH 7.4) (TM buffer) and once with 0.01 M TrisC1-0.01 M MgAcA.05 M KCl-0.01 M 0-mercaptoethano1 (pH 7.4) (TMKS buffer). The resultant cell paste was suspended in an equal volume of TMKS buffer, 4 pg/ mi of DNase (Worthington) was added, and the suspension was sonicated with a Branson sonifier. The suspension was clarified by at least two 30-min centrifugations at 30,OOOg and the final supernatant was centrifuged for 8 hr at 30,000 rpm in the No. 30 rotor of the Spinco Model L preparative ultracentrifuge. The upper twothirds of the supernatant (S-100) was aspirated, dialyzed against four changes of TMKS buffer, frozen, and stored at either -20 or -70". The ribosomal pellet was suspended in a buffer containing 0.01 M Tris-C1-0.002 M M g A c ~ 0 . 5M N H E l (pH 7.4) (Salas et al., 1965) with a glass-Teflon homogenizer. After standing for approximately 8 hr the suspension was centrifuged for 30 min at 30,OOOg (pellet discarded) and the ribosomes were pelleted by further centrifugation for 3 hr at 50,000 rpm
BODLEY
in the Spinco No. 50 Ti rotor. The supernatant was discarded and the dark brown upper layer of the pellet was scraped away and discarded prior to resuspending the ribosomes. This washing procedure was repeated twice and the ribosomes were finally suspended in a small volume of TM buffer. The suspension was dialyzed against four changes of TM buffer, centrifuged at low speed, adjusted to a concentration of 50 mg/ml, and stored at either - 20 or -70 ". In some cases these ribosomes were further purified as described by Kurland (1966), Le., by differential ammonium sulfate precipitation followed by further high-salt washing, or by chromatography on DEAE-cellulose (Stanley and Wahba, 1967). Except as is noted below, all of the preparations exhibited identical behavior with respect to the effect of heat. Ribosome concentrations were calculated assuming EiZ 14.5 (Tissikres et al., 1959). Although no functional alteration of ribosomes was detected as a result of repeated freezing and thawing, all data reported were derived with once frozen ribosomes. Amino acid incorporation experiments were typically performed in a final volume of 100 p1 and contained the following components: 100 mM Tris-C1 (pH 7.8), 50 mM KCl, 10 mM 0-mercaptoethanol, 5 mM phosphoenolpyruvate, 1 mM ATP, 0.05 m~ GTP, 30 pg/ml of pyruvate kinase, approximately 1 mg/ml of S-100 protein (typical S-100 preparations had protein concentrations, determined by the method of Lowry et al. (1951), of 10 mgirnl), 0.5 pCi/ml of [14C]amino acid (specific activities ranged from 248 to 366 mCi/mmole), 100 pg/ml of polynucleotide, up to 250 pg/ml of ribosomes, and optimal levels of MgAcz. Magnesium optima were determined for each preparation and were generally 15 mM for poly U directed incorporation and 14 mM for poly A. Slight differences, however, were noted in the optima for different preparations and all experiments were performed under optimal conditions. Usually a solution was made which contained all of the above components except ribosomes and the polynucleotide. Ribosomes, generally in a volume of 5 pl, were added and the reaction was initiated by the addition of 5 pl of an appropriate polynucleotide solution. Incubations were for 30-60 min at 37". Quantitation of the polypeptide product with poly U as the template was performed essentially by the method of Bollum (1966). Following incubation at 37 O, a portion of the reaction mixture (generally 75 pl) was removed and placed on a 2-cm square of Whatman No. 31ET paper. The paper was then immersed in 400-500 ml of 10% trichloroacetic acid. After all papers were collected (as many as 150) they were washed twice with 400-500-ml portions of 5 7, trichloroacetic acid and then heated at 90" for 15 min in 5 % trichloroacetic acid. The heating was followed by another wash in 5 7, trichloroacetic acid and then two washes each in ethanol-ether (l:l, v/v) and ether, respectively. All washes were for 5 min (except the one at 90") with gentle stirring while the papers were suspended in a wire basket. With poly A as the template the wash was the same except the papers were collected and washed twice in a solution of 5 Z trichloroacetic acid-0.25 7, sodium tungstate (pH 2.0) prior to heating at 90" in 57, trichloroacetic acid. After the final ether wash the papers were air dried and placed in
V O L . 8, N O . 2, F E B R U A R Y
1969
counting vials containing a toluene-based liquid scintillator (Packard Instrument Co.) and counted in a Beckman LS-100 scintillation counter. Counting efficiency under these conditions was approximately 70 %. Incorporation assays were linear for approximately 30 min and continued at a diminishing rate for 60-90 min. Theassays were linear with respect to ribosome concentration up to 250 pg/ml for the E. coli B system (some what less for the 413 system), the maximal level employed. Maximal incorporating activity (see below) for the B system was approximately 1.0 and 0.5 ppmole of amino acid per pg of ribosomes per 30 min for phenylalanine (poly U) and lysine (poly A), respectively. The 413 system was approximately three to four times as active, while the substitution of Q13 S-100forB S-100 approximately doubled the activity of B ribosomes. Radioactivity in the absence of either ribosomes or polynucleotide or with blank papers carried through the washing procedure generally did not exceed 100-200 cpm, and all of the data presented are corrected by such blank values. All incorporation data points, with the exception of the rate experiments, represent the average of at least duplicate determinations. The standard deviation of such replicates averaged < =t6 %. Sedimentation Velocity Analysis. Sedimentation analyses were performed with a Spinco Model E analytical ultracentrifuge equipped with schlieren optics. All runs were made with an AN-D rotor at a speed of 29,500 rpm using a 12-mm, 4" sector cell at a temperature of 22". The rotor was accelerated at a constant rate (12 A) and timing was begun upon reaching a speed of 20,000 rpm (which required ~ 2 . min). 6 The rotor was near speed approximately 1.2 min later. Unless otherwise specified, the sedimentation profiles were made from photographs taken at either 24 or 40 min with a schlieren bar angle of 60". All sedimentation analyses were performed at a ribosome concentration of 5 mg/ml in a volume of 0.7 ml. The graphically depicted sedimentation coefficients are uncorrected for ribosome concentration or buffer salts and assume an average minisciis distance from the center of rotation of 5.98 cm. With the particular enlargement employed, the distance between the two outer boundaries of the sedimentation profile figures is proportional to the distance between the counter weight reference holes (1.6 cm) in the rotor. The relative proportion of the various ribosomal components was determined by measuring the areas of the schlieren peaks. Thermal denaturation was conducted in a rapidly circulating water bath maintained to within h O . 1 " of the nominal temperature. Most of the experiments were conducted under conditions where the sample temperature (as measured with a thermistor probe) was within 1O of the bath temperature within 20 sec and was indiscernably different within 30 sec. Heating was terminated by either immersing the tube in an ice bath or removing an aliquot and adding it to an ice-cold assay solution. The two methods yielded identical results in terms of the loss of biological activity. Unless otherwise stated all heating experiments were performed at a ribosome concentration of 5 mg/ml. As indicated above, ribosomes were stored at a con-
*'
*\
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--
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*, 10
20
30
TIME (MINI FIGURE 1: Rate of thermal inactivation of ribosomes in TM buffer. Separate but identical solutions of E. coli B ribosomes were heated at the indicated temperatures. Aliquots were periodically withdrawn and added to an assay solution and subsequently assayed for their ability to incorporate phenylalanine in response to poly U. Activity is expressed as per cent of that exhibited by unheated ribosomes. Procedural details in this and subsequent figures are as outlined in Materials and Methods.
centration of 50 mg/ml in TM buffer. In order to add salt, the ribosomes were simply diluted into the appropriate buffer. In order to reduce the magnesium concentration the ribosomes were first diluted with low magnesium buffer and then dialyzed 12-24 hr against this buffer. Optical Measurements. All optical measurements were performed at ambient temperature in a Beckman DU spectrophotometer equipped with IO-mm microcells. Turbidity measurements of both native and denatured ribosomes (5 mg/ml) followed the relationship of Bayzer and Schauenstein (1955) over the range 400-700 mp, namely, that a plot of log OD vs. log X was linear, indicating the contribution of light scattering rather than chromophore adsorption. By extrapolation of such plots it was possible to show that with an A400 of 1.0 the contribution of turbidity to absorbancy at 260 mp would be less than 10%. Results
Effect of Heating on Biological Activity. Figure 1 illustrates a typical experiment in which the rate of loss of the biological activity of ribosomes (the ability to participate in the formation of polyphenylalanine) was followed as a function of time of incubation at various temperatures in TM buffer. The first effect observed
467
THERMAL DENATURATION OF RIBOSOMES
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upon heating, although not marked with this ribosome preparation (see below), was an initial increase in the ability to support phenylalanine incorporation. Activity is then lost at a rapid but decreasing rate. Ribosomes did not exhibit first-order denaturation kinetics under any of the conditions examined. Perhaps the most striking feature of these data is the narrow range of temperature over which denaturation occurs. In fact, experiments have shown that at 50" in TM buffer approximately 6.5 hr is required for a 50z decrease in polyphenylalanine-synthesizing ability. The abruptness of this transition is more clearly demonstrated in Figure 2 (middle curve). These data were generated by heating separate but identical solutions of ribosomes for 5 min at various temperatures and, after cooling, measuring their ability to translate poly U. For convenience, the temperature at which 50 inactivation occurred was designated as the Td,5. The average Td,6for ribosomes in TM buffer was 57". Raising the level of magnesium in this buffer did not enhance the stability of the particles. Denaturation profiles are also shown in Figure 2 for ribosomes dissolved in two other commonly employed buffers, TM10-4 buffer (0.01 M Tri~-Cl-lO-~MMgAcs, pH 7.4) and TMK buffer (0.01 M Tris-C1-0.01 M MgAcz-O.l M KC1, pH 7.4). Ribosomes in TMlO-4 buffer and TMK buffer exhibited a Td.5 of 53.5 and 60.5", respectively. The values of Td,, obtained by graphic interpolation were surprisingly constant with a maximum deviation generally less than k0.5"and the behavior of the ribosomes was not altered by the further purification outlined in Materials and Methods. Out of six ribosome preparations (three E . coli B and three 413) only one showed significantly different thermal stability with averaging approximately 1-2" lower in values of Td,5 the buffers tested. Perhaps coincidentally, thissamepreparation also exhibited the maximum increase in activity on heating (Figures 4 and 5). The and Td.80in TM and TMK buffers are 1 and 3" lower than the respective Td.5'S (data not shown). Experiments have shown the Td,5to be essentially independent of ribosome concentration (during heating) between 0.5 and 50 mg per ml in TM buffer. Experiments have also shown that the denaturation rate in TM buffer is essentially independent of pH over the range pH 7-8 and only slightly greater at pH 9. While it is known (Good et al., 1966) that the pH of Tris-C1 buffer is strongly temperature dependent, it is unlikely that the present observations reflect a pH effect as such. In addition, P-mercaptoethanol(1-10 mM) had no detectable effect on thermal stability at least upon short-term incubation. It should also be pointed out that the rate of thermal destruction of ribosomal activity was not noticeably affected by the composition of the subsequent assay mixture (at least in relation to the source of the S-100, B or Q13, or the concentration of amino acid, polynucleotide, or tRNA, or the length of the incorporation incubation). Clearly, however, the absolute activities varied widely under these conditions. It is quite clear from the data in Figure 2 that KCl serves to protect the ribosome from thermal inactivation. This protection by salt is further explored in
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468
BODLEY
TEMP ("C) Thermal destruction of biological activity as a function of buffer composition. Separate solutions of ribosomes in the indicated buffers were heated for 5 min at various temperatures, chilled, and assayed for polyphenylalaninesynthesizing ability.
FIGURE 2:
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LOG SALT CONC (MI FIGURE 3: Protection from thermal inactivation by monovalent cations. Ribosomes in TM buffer supplemented with the indicated salt concentration were heated for 20 min at 60", chilled, and assayed. The activity (polyphenylalanine synthesis)is expressed as per cent of the unheated ribosomes. The level of activity remaining after heating in TM buffer alone (0.8 %) is indicated by the broken line.
Figure 3. It is evident that maximum stability is achieved in the presence of 0.1 M KCl while N H E l and NaCl are, in that order, less effective in protecting the ribosome against thermal inactivation. With one preparation of ribosomes N H C l at a somewhat higher concentration was approximately as protective as KCl. As was previously noted, one of the distinguishing results of heating ribosome solutions was an initial increase in activity at moderate temperatures. Figure 4
8,
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F E B R U A R Y
1969 I
2.5 I h
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TIME (MINI
TEMP PC) FIGURE 4: The effect of 5 rnin incubation in TMK buffer at various temperatures on the ability of ribosomes to translate poly U and poly A. After heating at the indicated temperatures the solutions were chilled and added to the assay solutions optimized for the three activities. The assay incubation was for 60 rnin at 37". The results are expressed in ppmoles of amino acid incorporated per 100 p1 of assay (25 pg of ribosomes).
illustrates this phenomenon with a ribosome preparation which showed a more pronounced increase. With this preparation heating for 5 min a t 50" doubled the ability of the ribosomes to support polyphenylalanine synthesis and increased their ability to translate poly A by sixfold. This thermal "activation" varied with different ribosome preparations from 0 to 100% for poly U translation and 0-500 2 for poly A translation, but no correlation was evident between thermally induced increases in the ability to translate these two polynucleotides. I t was evident, however, that the thermally "activated" ribosomes possessed activities which were more comparable than ribosomes which had not been heated. Figures 4 and 5 also illustrate that the leucine ambiguity (see Bodley and Davie, 1966) increased in parallel with phenylalanine incorporation. Figure 5 also shows that the rate of loss of activity with poly U (both phenylalanine and leucine incorporation) or poly A as messenger was identical. Thermally Induced Physical Changes. Figure 6 illustrates the turbidity of ribosome solutions at ambient temperature after heating for 5 rnin at various tempertures in the three buffers. The turbidity of unheated ribosome solutions a t this concentration (5 mg/ml, 72.5) is low and ribosomes in TMlOW4buffer routinely exhibited one-third to one-half the turbidity of ribosomes in TM buffer. Heating for 5 rnin in this latter buffer resulted in a nearly linear A400increase over the temperature range 52-62' and then rose sharply. This sharp rise in turbidity presumably reflects either aggregation of ribosomes or coagulation of ribosomal protein. I n low magnesium (TMlO-4 buffer) there was no significant change in turbidity up to 58". Readdition of
FIGURE 5 : Rate of loss of poly U and poly A translating ability upon incubation of ribosomes in TMK buffer at 58". A ribosome solution was placed in a bath at 58' and samples were periodically withdrawn and added to cold assay solutions for subsequent measurements of incorporating ability. Activity is expressed as per cent of the unheated controls. (a)Lysine incorporation with poly A as template; (0) phenylalanine; and (D) leucine incorporation with poly U as template.
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60
65
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TEMP PC) 6: Effect of heating on ribosomal turbidity in various buffers. Ribosomes in the indicated buffers were heated 5 min at the indicated temperature and chilled, and their Aaoa was determined at ambient temperature. The x represents the turbidity of ribosome solutions which have been made 0.01 M in MgAcz after heating in TM10-4 buffer. FIGURE
magnesium to ribosomes heated in TM10-4 buffer increased the turbidity by the difference between unheated ribosomes in T M and TM10-4 buffers, respectively. The turbidity in TMK buffer rose gradually till approximately 62 '. The nature of the physical change of ribosomes following heating in T M buffer is recorded in Figure 7a. There was no major change in the qualitative nature of the sedimentation profile but simply a loss of material sedimenting as 100 S or less. This material appeared
T H E R M A L
D E N A T U R A T I O N
469
OF RIBOSOMES
BIOCHEMISTRY
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TEMP ('C) 7: Sedimentation behavior of ribosomes heated in TM buffer. (a) Sedimentation profiles of ribosomes after heating lor 5 min at the indicated temperatures. The m in this and subsequent figures marks the positions of the menisci. (b) The data points represent the per cent (relative to the unheated control) particles sedimenting 100 S. The broken lines are schematic representations oftheactivity(Figure 2 ) and turbidity (Figure 5 ) under theseconditions. FIGURE
