Ribosomal Proteins of Escherichia coli*

Proceedings of the National Academy of Sciences Vol. 67, No. 4, pp. 1909-1913, December 1970 Ribosomal Proteins, XIII. Molecular Weights of Isolated Ribosomal Proteins of Escherichia coli* M. Dzionara, E. Kaltschmidt, and H. G. Wittmann MAX-PLANCK-INSTITUT FtR MOLEKULARE GENETIK, BERLIN-DAHLEM, GERMAMY Communicated by Severo Ochoa, September 23, 1970 Abstract. M\lolecular weights of the isolated proteins from the 30S and 50S ribosomal subunits of Escherichia coli were determined by two independent methods: polyacrylamide gel electrophoresis with sodium dodecylsulphate and equilibrium sedimentation. The values for the molecular weights determined by gel electrophoresis range from 10,900 to 65,000 for the proteins of the 30S subunit and from 9,600 to 31,500 for those of the 50S subunit, with number averages of 19,000 and 16,300, respectively, in agreement with those obtained by equilibrium sedimentation. The ribosomes of Escherichia coli consist of a number of different proteins,' some of which have been isolated and characterized.2-8 According to recent studies there are about 55 individual proteins in the 70S ribosomes of E. coli.9 l0 The molecular weights of some 20 proteins isolated from the 30S subunit have been determined by equilibrium sedimentation.8 Furthermore, molecular weights have been determined, by electrophoresis in sodium dodecyl sulfate (SDS)-containing polyacrylamide gels, for partially fractionated ribosomal proteins.9 The present paper describes the determination of the molecular weights of the individual ribosomal proteins11-'3 by two independent methods. Materials and Methods. The ribosomal proteins of E. coli K12, strain A19, were isolated as described."-'3 All were homogeneous as judged by one- and two-dimensional polyacrylamide gel electrophoresis. These proteins were also found to be homogeneous (unpublished data) when tested by cellulose acetate electrophoresis.14 SDS-polyacrylamide gel electrophoresis was conducted essentially by the procedure of Weber and Osborn."5 However, the gels were not polymerized in columns but in slabs. Samples and a bromophenol-blue marker were applied to small slits in the gel. The bromophenol blue mobility was taken as a standard; all protein migrations were normalized to this value. Using the two-dimensional apparatus of Kaltschmidt and Wittmann,'6 it was possible to simultaneously run five gel-slabs (a total of 75 samples); thus, identical conditions were created for all of the ribosomal proteins in one run. Electrophoresis was usually for 17 hr at 1.5 V/cm. Each gel slab was then removed from its individual frame and the distance the bromophenol blue band had traveled was marked. Proteins were stained with Coomassie brilliant blue. The calibration was performed with the following proteins: Cytochrome c (molecular weight 11,700); pancreatic ribonuclease (13,700); lysozyme (14,300); hemoglobin, a- chain (15,500); myoglobin (17,200); TMV-coat protein (17,500); -y-globulin, L-chain (23,000); trypsinogen (24,000); chymotrypsinogen (25,700); elastase (25,900); pepsin (35,000); glyceraldehydephosphate dehydrogenase (36,000); bovine serum albumin (68,000). The marker proteins moved in the same way as described by Weber and Osborn,'5 except that we observed a somewhat greater deviation from the calibration curve. 1909

1910 BIOCHEMISTRY: DZIONARA ET AL. PROC. N. A. S. The average deviation was about 15%. We suspected that incomplete denaturation during treatment of the samples (2 hr at 370C) in 0.01 M sodium phosphate buffer (ph 7, 1% SDS, 1% #-mercaptoethanol, 8 M urea) was responsible for this deviation. Therefore, RNase, with the most deviant mobility, and myoglobin and glyceraldehydephosphate dehydrogenase, both of which showed normal behavior, were incubated for 30, 60, 120, and 240 min; this treatment did not affect the mobilities. In each of the five slabs of gel in an experiment, the four marker proteins were run which best fitted the calibration curve; they were lysozyme, myoglobin, chymotrypsinogen, and glyceraldehydephosphate dehydrogenase. Ultracentrifugation: Molecular weights were determined by the high speed equilibrium sedimentation method of Yphantis'7 in a Spinco model E analytical ultracentrifuge equipped with electronic speed control and interference optics. The method of Nazarian'8 was applied when the concentration condition of c = 0 at the meniscus was not satisfied. Centrifugation was at 40,000 rpm for 22 hr at 20'C. Interference fringes were measured with a Leitz traveling microscope. All samples were run in 10% acetic acid, 0.2 M sodium chloride, 6 M urea, and 0.1%,3-mercaptoethanol. The density of this solvent, as determined in a pycnometer, was 1.111 g/cm3. Partial specific volumes were calculated from the amino acid compositions. The lyophilized proteins were dissolved in the above solvent and dialyzed against it overnight. Proteins, which after column chromatography had been stored at - 15 C in 10% acetic acid, were concentrated in a dialysis bag by evaporation of the acetic acid, and subsequently dialyzed exhaustively against the solvent. The concentration of the proteins ranged from 0.005 to 1 mg/ml. Lysozyme and ribonuclease, which were run for comparison, gave data that yielded straight line plots of ln c against r2/2, and the calculated molecular weights were within 8% of the value computed from the amino acid sequences. Molecular weights given in Results are those from experiments in which the corresponding proteins yielded straight plots or at least long straight segments. Results. Fig. 1 shows a gel slab with the four calibration proteins and nine ribosomal proteins. With the ribosomal proteins, one occasionally sees a major and some minor bands as in the fourth sample from the right. The minor bands probably result from aggregation of the proteins in the main bands because their molecular weights correspond to dimers, trimers, etc., of the main component (Fig. 2). Tables 1 (first three columns) and 2 show the molecular weights of the 30S and 50S proteins determined in this study. The agreement between molecular weights determined by gel electrophoresis and sedimentation is in most cases within the limits of experimental error. Discussion. The molecular weights of the proteins from the 30S particle, determined by the SDS-gel method, lie between 10,900 and 65,000, and those from the 50S subunit between 9,600 and 31,500. The sum of the molecular weights of the proteins of the smaller subunit is 380,000 and those of the larger subunit 555,000, with number average molecular weights of 19,000 and 16,300, respectively. Fig. 3 is a histogram of the distribution of the proteins, in groups of 1,000 daltons, for both subunits. By immunological and electrophoretic studies (unpublished data), it was possible to correlate our protein bands directly with those reported by Kurland's group and indirectly8 also to those of Tissieres' group. Table 1 shows a comparison of the molecular weights of the 30S ribosomal proteins determined in the three laboratories. They are in relatively good agreement. Our proteins L7 and L12 (Table 2) are identical to the proteins Al and A2 isblatedfby Mbller et al.,19 for which they reported molecular

VOL. 67, 1970 RIBOSOMAL PROTEINS 1911 Molecular weights LI---. * JL;iS4 *- 50,600 -Nbi-< 38,700 IMX 4-..- 25,900 * 4-- 12,400 (Left) FIG. 1. SDS-gel slab with four calibration proteins and nine ribosomal proteins (from left to right). The marker proteins are chymotrypsinogen, lysozyme, myoglobin, and glyceraldehydephosphate dehydrogenase. (Right) FIG. 2. SDS-gel electrophoretic pattern of a ribosomal protein, showing monomer and probable di-, tri-, and tetramers; the molecular weights are indicated. TABLE 1. Comparison of molecular weights of 30S ribosomal proteins determined in three laboratories. Protein Protein (Madison, Protein (Berlin Equil. Kurland8 Equil. (Geneva code) SDS* sed.t code) sed.t Chem.1 code) SDS* Si 65,000(2) Notdone 1 65,000 31,000 13 68,000 S2 28,300(4) 24,000 4 a 30,000 27,300 S3 28,200(8) 23,000 9 33,000 14,200 10 b 29,900 S4 26,700(3) 23,000 10 26,700 19,300 S5 19,600(8) 18,500 3 24,000 16,200 8 a 20,200 S6 15,600(6) 15,500 2 18,000 18,000 10 a 13,500 S7 22,700(3) 26,000 S8 15,500(5) 15,500 2 a 17,600 17,600 11 29,800 S9 16,200(16) 14,500 12 21,000 13,500 5 16,200 Slo 12,400(2) 18,000 4 16,000 14,800 6 10,500 S12 17,200(5) 15,000 5 19,000 16,000 S13 14,900(4) 14,000 15 S14 14,000(3) 14,000 12 b 15,600 14,200 S15 12,500(4) 13,000 14 13,200 15,800 4 b 10,700 S16 11,700(5) 13,000 6 13,500 11,700 3 9,800/12,900 S17 10,900(1) Notdone 7 10,700 15,600 4 a 9,600 S18 12,200(5) 10,500 12 a 14,600 11,000 S19 13,100(3) 14,000 13 15,000 13,000 2 a 11,400 S20 12,000(6) 12,500 16 14,000 12,000 1 10,800 S21 12,200(4) 13,500 15 a 13,000 15,700 * Electrophoresis in polyacylamide gels with sodium dodecyl sulfate used to determine molecular weight. Figures in parentheses give the number of independent protein isolations tested. t Molecular weight determined by centrifugation. T Molecular weight determined by chemical analysis and amino acid content.

1912 BIOCHEMISTRY: DZIONARA ET AL. PROC. N. A. S. TABLE 2. Molecular weihts of 50S ribosomal proteins of E. coli K. Molecjvr weight Molecular weight Protein SDS* Sedimentationt Protein SDS* Sedimentationt Li 26,700(1) 22,000 L18 14,300 (1) 17,000 L2 31,500(2) 28,000 L19 14,900 (4) 17,500 L3 27,000 (4) 23,000 L20 17,200 (4) 16,000 L4 25,800 (3) 28,500 L21 13,900 (5) 14,000 L5 22,000 (2) 17,500 L22 14,800 (2) 17,000 L6 22,200 (4) 21,000 L23 12,700 (3) 12,500 L7 13,400 (3) 15,500 L24 14,300 (12) 17,500 L8 17,300 (4) 19,000 L25 12,000 (3) 12,500 L9 17,300(1) Not determined L26 12,000 (2) 12,500 L10 19,000 (4) 21,000 L27 12,700 (1) 12,000 L11 19,600 (3) 19,000 L28 12,300 (2) 15,000 L12 13,200 (1) 15,500 L29 12,000 (1) 12,000 L13 17,800 (1) 20,000 L30 11,200 (5) 10,000 L14 16,200 (6) 18,500 L31 10,000(1) Not determined L15 17,500 (4) 17,000 L32 10,500(1) Not determined L16 17,900 (4) 22,000 L33 10,500 (2) 9,000- L17 16,700 (8) 15,000 L34 9,600(1) Not determined * Determined by SDS-gel electrophoresis. Number of separate isolations in parentheses. t Determined by sedimentation equilibrium. NO. OF PROTEINS NO. OF PROTEINS 30S 5 50S FIG. 3. 1 2 3 6.5 MW1/ 04 1 2 3 MW/10i Number of proteins plotted against their molecular weights, as determined by SDS gel electrophoresis, in groups of 1000 daltons. weights of about 18,000. However, in recent studies (M6ller, personal communication) molecular weights of about 15,000 have been assigned to these proteins. Hill et al.20 determined the molecular weight of the 30S subunit to be 900,000 ± 30,000. Its protein content was estimated to be about 37% by Tissi~res et al.,21 and 30-33% by Craven et al.5 Thus, the total molecular weight of the ribosomal proteins must lie within the range of 260,000-345,000. The average molecular weight (302,000) of the total 30S proteins thus calculated is less than the sum of the molecular weights of the individual proteins, namely, 380,000. This result supports the hypothesis of a population of heterogeneous proteins in the 30S subunits proposed by Kurland and co-workers.8'22 A similar calculation for the 50S subunit, with a molecular weight of 1.55 4 0.05 X 106 (ref. 20) and a protein content of 37 : 3%, results in a range of 510,000-640,000 for the molecular weight of the proteins of the 50S subunit, in good agreement with the sum of the molecular weights for the individual proteins, namely, 555,000. On the basis of these results, the 50S subunits are more likely to be homogeneous than the 30S subunits.

VOL. 67, 1970 RIBOSOMAL PROTEINS 1913 We wish to thank Miss J. Huf and Mr. A. Franz for excellent technical assistance. was supported by the Deutsche Forschungsgemeinschaft. Abbreviation: SDS, sodium dodecyl sulfate. This work * Previous paper of this series: Kaltschmidt, E., and H. G. Wittmann, Proc. Nat. Acad. Sci. USA, 67, 1276 (1960). 1 Waller, J. P., J. Mol. Biol., 10, 315 (1964). 2 Traut, R. R., P. B. Moore, H. Delius, H. Noller, and A. Tissieres, Proc. Nat. Acad. Sci. USA, 57, 1294 (1967). 8 Kaltschmidt, E., M. Dzionara, D. Donner, and H. G. Wittmann, Mol. Gen. Genet., 100, 364 (1967). 4M6ller, W., and J. Widdowson, J. Mol. Biol., 24, 364 (1967). 5 Fogel, S., and P. S. Sypherd, Proc. Nat. Acad. Sci. USA, 59, 1329 (1968). 6 Moore, P. B., R. R. Traut, H. Noller, P. Pearson, and H. Delius, J. Mol. Biol., 31, 441 (1968). Hardy, S. J. S., C. G. Kurland, P. Voynow, and G. Mora, Biochemistry, 8, 2897 (1969). 8 Craven, G., P. Voynow, S. J. S. Hardy, and C. G. Kurland, Biochemistry, 8, 2906 (1969). 9 Traut, R. R., H. Delius, C. Ahmad-Zadeh, T. A. Bickle, P. Pearson, and A. Tissibres, Cold Spring Harbor Symp. Quant. Biol., 34, 25 (1969). 10 Kaltschmidt, E., and H. G. Wittmann, Proc. Nat. Acad. Sci. USA, 67, 1276 (1970). 11 Hindennach, J., G. St6ffler, and H. G. Wittmann, submitted for publication. (Eur. J. Biochem.) 12 Hindennach, J., E. Kaltschmidt, and H. G. Wittmann, submitted for publication. (Eur. J. Biochem.) 13 Kaltschmidt, E., V. Rudloff, H. G. Janda, M. Cech, K. Nierhaus, and H. G. Wittmann, submitted for publication. (Eur. J. Biochem.) 14 St6ffler, G., Mol. Gen. Genet., 100, 374 (1967). 15 Weber, K., and M. Osborn, J. Biol. Chem., 244, 4406 (1969). 16 Kaltschmidt, E., and H. G. Wittmann, Anal. Biochem., 36, 401 (1970). 17 Yphantis, D. A., Biochemistry, 3, 297 (1964). 18 Nazarian, G. M., Anal. Chem., 40, 1766 (1968). 19 Moller, W., H. Castleman, and C. P. Terhorst, FEBS Lett., 8, 192 (1970). 20 Hill, W. E., G. P. Rossetti, and K. E. v. Holde, J. Mol. Biol., 55, 263 (1969). 21 Tissibres, A., D. Schlessinger, and B. R. Hollingworth, J. Mol. Biol., 1, 221 (1959). 22 Kurland, C. G., P. Voynow, S. J. S. Hardy, L. Randall, and L. Lutter, Cold Spring Harbor Symp. Quant. Biol., 34, 17 (1969).