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Composition of ribosomes of an extremely halophilic bacterium
J. Mol. Biol. (1966) 15,420-427
Composition of Ribosomes of an Extremely Halophilic Bacterium S. T.
BAYLEY
Dioieum. of Biosciencee National Beseardi Council Ottawa, Ontario, Canada (Received 15 September 1965) Amino acid analyses and electrophoresis on starch-urea gels have shown that the ribosomes of the extremely halophilic bacterium, Halobacterium cutirubrum, contain a majority of acidic proteins with isoelectric points of about 3·9 and a. smaller proportion of basic proteins with isoelectric points above about 8·5. From analyses of magnesium and potassium, it is concluded that magnesium ions probably stabilize the RNA moiety as in other ribosomes, and that potassium ions neutralize negative charges, thereby enabling the acidic proteins to be bound to the ribosomal RNA by hydrogen and hydrophobic bonds. The acidic and basic proteins are similar in amino acid composition except for the relative proportions of acidic to basic residues. The possible significance of this observation in relation to halophilism is discussed.
1. Introduction In ribosomes from a wide variety of sources such as E8cherichia coli (Waller & Harris, 1961; Spahr, 1962; Waller, 1964), pea seedlings (Setterfield, Neelin, Neelin & Bayley, 1960), rat liver (Crampton & Petermann, 1959) and rabbit reticulocytes (Cohn, 1962; Mathias & Williamson, 1964), the structural proteins are predominantly basic and appear to be bound to the phosphate groups of the RNA (Edelman, Ts'o & Vinograd, 1960). An exception to this general pattern was found recently in the extremely halophilic bacterium, Halobacterium cutirubrum (Bayley & Kushner, 1964). The ribosomes from this organism are stable as 70 s particles in 4 M-KCl; 0·1 M-MgCl2 and 0·01 M-tris buffer (pH 7'2). In 0·005 M-tris with 0·005 y-MgCl 2 , roughly two-thirds of the structural protein dissolves to leave RNA-rich particles which sediment at rates similar to those of the RNA moieties of E. coli ribosomes (Bayley & Kushner, 1964; Moller, 1964). Electrophoretic studies showed that the great majority of the dissolved proteins were acidic, with isoelectric points of about 3·9. It is believed that these proteins remain associated with the negatively charged RNA only so long as the negative charges in the complex are shielded by suitable concentrations of cations. To investigate the ribosomal proteins of this halophile further, amino acid analyses have been carried out on whole ribosomes, on the protein which dissolves at low salt concentrations and on the RNA-rich particles which remain. Conclusions reached from these analyses have been confirmed by electrophoresis of the proteins on starch-urea gels. To examine the manner in which these proteins are bound to the ribosomal RNA, the Mg and K contents of ribosomal particles have also been estimated. 420
COMPOSITION OF HALOPHILE RIBOSOMES
421
2. Materials and Methods Ribosomes from H. cutirubrum were prepared in 4 M-KCI; 0·1 M-MgCI:a and 0·01 M-tris buffer (pH 7,2) (solution D), and purified by 2 cycles of differential centrifugation as previously described (Bayley & Kushner, 1964), except that the harvested, washed cells were ground with 1 to 1·5 times their wet weight of alumina, instead of being shaken with ballotini beads. (a) Amino acid analyses Two separate sets of ribosomal protein samples were prepared and analysed. In each experiment, the final ribosomal suspension was centrifuged at 50,000 rev.fmin for 90 min and the ribosomal pellet was resuspended in 0·005 M-tris-MgCI 2 (0,005 M-TMg) (experiment 1) or 0·005 M-tris-MgCI 2-KCI (0,005 M-TMgK) (experiment 2) to a final concentration of about 1 %. A sample of this suspension, representing whole ribosomes, was kept for analysis, while the remainder was centrifuged at 50,000 rev.fmin for 180 min to sediment the RNA-rich particles. The supernatant fraction, containing the soluble protein, was kept for analysis, while the pellet was resuspended in fresh low-salt buffer. The suspension was centrifuged at 50,000 rev.fmin for 180 min and the final pellet, after resuspension in fresh buffer, was analysed as the RNA-rich particles. All samples were freeze-dried. For the amino acid analyses, two portions of each sample, each containing roughly 4 mg protein, were digested after the method of Moore & Stein (1963) for periods of 20 hr and 70 hr, respectively. Because of their high RNA contents, the samples of whole ribosomes and of RNA-rich particles showed extensive humin formation. These were therefore centrifuged or filtered after hydrolysis to remove the deposits. Analyses were carried out on a Beckman Spinco model 120B amino acid analyser. In experiment 1 alone, the analyses were duplicated and agreed closely.
(b) Other chemical analyses Samples to be analysed for Mg and K were prepared as follows. A ribosomal suspension in solution D was centrifuged at 50,000 rev.fmin for 90 min. The supernatant solution was discarded and the ribosomal pellet and the walls of the tube were carefully rinsed twice with 0·005 M-TMgK to remove excess D. The pellet was then resuspended in 0'005 M-TMgK to yield whole ribosomes. This suspension was then centrifuged at 50,000 rev.f min for 180 min to yield a pellet, part of which was resuspended in water and repre· sented RNA-rich particles 1. The remainder was resuspended in 0·005 M-TMgK and centrifuged as before. The resulting pellet was resuspended in water to yield RNA-rich particles 2. Potassium was estimated with a Beckman flame spectrophotometer. To avoid interference from phosphorus and other cations, Mg was estimated from t.ho turbidity formed on adding potassium erucicate, after the method of Marier & Boulet (1959). Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951), using plasma albumin as a standard, and phosphorus by the method of Allen (1940). The weight of RNA was taken as 11 times the weight of phosphorus. (c) Gel electrophoresis Samples of whole ribosomes and of RNA-rich particles were prepared in the same manner as for amino acid analyses, except that the final suspension in each case was in pH 5·2 acetate or pH 8·0 veronal buffer of ionic strength 0·02 at a concentration of about 14 mg protein/nil. Before electrophoresis, 420 mg urea was added to each 1 ml. of sample. To digest the ribosomal RNA so as to release the protein for electrophoresis, a solution of pancreatic RNase (Worthington), pretreated to destroy proteolytic activity (Swift, 1955), was then added, after the method of Waller (1964), to give a concentration of roughly 1 p,g RNasefmg RNA in the sample. Electrophoresis was carried out horizontally in starchurea gels as previously described (Bayley & Kushner, 1964).
422
S.T. BAYLEY
3. Results (a) Amino acid analyses
In the two separate preparations analysed, the ratios of RNA to protein for whole ribosomes were l·ll and 1'36; for soluble protein, 0·051 and 0·1l6; and for RNA-rich particles, 3·4 and 4'09, respectively. In each case, the soluble protein fraction accounted for 60 to 65%, and the RNA-rich particles for about 30%, of the total ribosomal protein. The results of the amino acid analyses for the two preparations are given in Table 1. With the exceptions noted, the values quoted are from the 20-hour hydrolysates. In the analyses of the RNA-rich particles, a measurable peak occurred between alanine and valine, whether the 50°C or the 30 and 50°C programme (Zacharius & Talley, 1962) was used on the column. This material was therefore not cystine, which in the latter programme should be eluted after valine (Zaoharius & Talley, 1962), but it has not been examined further. With whole ribosomes and RNA-rich particles, values have been omitted for ammonia and glycine because of the production of these substances from RNA. For soluble proteins, the ammonia value is taken as an approximate measure of amide nitrogen (Moore & Stein, 1963). The total weight of all the components listed per 100 g soluble protein was estimated to be 70·0 g and 110·7 g in the two experiments, respectively. As a comparison, Table 1 includes a less accurate analysis of the proteins in isolated cell envelopes of H. cutirubrum (Kushner, Bayley, Boring, Kates & Gibbons, 1964), as well as the thorough analysis by Spahr (1962) of the protein from 70 s E. coli ribosomes. In content of neutral residues, there are significant differences between the proteins listed in Table 1, although, for the soluble proteins and the proteins associated with the RNA-rich particles of H. cutirubrum, these differences are small. The distinctive feature of most of the halophile proteins is the high content of acidic, and the relatively low content of basic, amino acids. The excess of aspartic and glutamic acids, less amide ammonia, over lysine, histidine and arginine is 7·6 moles/lOOmoles in the soluble ribosomal proteins, exceeded only by the envelope protein with 11·5. By contrast, the ribosomal proteins of E. coli have an excess of basic over acidic amino acids of 7·6 moles %. (This differs from the figure given by Spahr (1962), because some of the recovered residues have been omitted from the total considered here.) The excess of acidic residues in the soluble ribosomal proteins supports the electrophoretic evidence for the acidic nature of these proteins (Bayley & Kushner, 1964). The ratios of basic to acidic residues (Table 1) suggest that the proteins which remain associated with the RNA in the RNA-rich particles are considerably more basic than those that dissolve. However, it is evident from the results of gel electrophoresis which follow that the analysis for RNA-rich particles actually represents an average of both acidic and basic proteins. (b) Gel electrophoresis
To examine the basicity of the proteins of the RNA-rich particles further, samples of whole ribosomes and of RNA-rich particles were subjected to electrophoresis in starch-urea gels in the presence of RNase (Waller, 1964) as described above. The patterns in Plate I, together with that obtained from whole ribosomes in veronal buffer (not shown), clearly demonstrate that these ribosomes contain proteins with an isoelectric point above about 8·5. As is to be expected from the extensive loss of
(a)
(b)
(c)
PLATE 1. (a) (b) (c) Electrophoretic p atterns in starch-urea gel sta ined with n ig ro s in , aft er 6 hr at i rn A/gel , of (a) whol e H . cutirubrum ribosomes and (b ), (e) R X A-r ieh p articles, all treated overn ight wi th R'Nase in urea . The gels wer e made up (a ) a nd (b ) in aceta te buffer (pH 5,2) a nd (c ) in ver orial b uffer (p H 8 '0 ), a lt ho ug h the pH val ues in the gels were 0·3 t o 0·;; unit h ig her. The anode is at the bottom. R esu lt s wit h amid o black stainin g we re simi la r . On an RNA b a sis, the quantities of RNA-rich part icles in (b) and (c) wer e 1·5 times tha t of whole ribosomes in (a). [facing p . i t :!
TABLE
1
Amino acid analyses, expressed as moles/100 moles amino acids recovered, for (a ) soluble ribosomal protein, (b) RNA-rich particles, and (c) whole ribosomes from H. cutirubrum compared to (d) envelope protein of H . cutirubrum, and (e) 70 s E. coli ribosomal protein Amino ac id
Meana Lysine Histidine Ammonia b • d Arginine Aspartic acid Threonine" Serine> Glutamic acid Proline GlycineAlanine Half-eyst.ino'[ Valine Methionine Isoleucine Leucine Tyrosino'' Phenylalanine Ratio
a b
o d 8
Lys
+ His + Arg + Asp
GIu
3·6 : 2·3 ; (8'{ ; 6·4 ; 14·6;
3'{ 2·0 12' 7) 6·5 14 ·9
{'O ;
{j08
{ 'O :
15 ·1 ; 4·3 ; (9'1 ; 12·0:
4·0 16·5 4·2 10'3 ) 13·0
10.2°: 1·5 : 4'5° ; 7·4 ; 2·2 ; 2·2;
10·6 1·3 5·1° 7·9 2·0 2·3
Whole H. cutirubru m ribosomes
RNA-rich particles
Soluble protein
3·7 2·1 (10'7) 6·4 14·7 6·4 5·5 15·8 4·3 (9'7) 12·5 0·0 10·4 1·4 4·8 7·6 2·1 2·3
6·5 2·2
4·9 ; 4·0 2·2 : 2·1
10·3; 11'6; {'6 ; 7'{ ; 11·8; 5·0 ;
10·6 13 '{ 5·3 3·2 13·6 5·3
10·5 12,7 6 ·5 5·4 12·7 5· 1
13 ·3 ; 12·9
13·1 tr? 9·4 1·3 4·6 6·4 1·4 2·2
{'6 ; 13 ·4 : {'1 : {'8 ; 14,2; 4'6 ;
6·8 13 '{ {'{
7·6 15 ·3 4·5
8·8°; 1·3 : 4'2°: 6·0 ; 2·0 ; 2'1;
9·9° 1·2 5.0 0 6·8 0·8 2·4
0 ·4
Mean of the result s from experimen t s 1 and 2 shown in italics. Extrapolated to zero hydrolysis time (Moore & Stein, 1963 ). Value for 70-hr hydrolysate (Moore & Stein, 1963: Spahr, 1962). Not included in total. From Kushner et al, ( 1964). From Spahr (1962); 0. small amount. of tryptophan was also found.
0·76
70 s E . coli ribosomal protein!
Mean a
Mean a
6·3 : 6·9 2·0 : 2·4
Envelope protein from H. cutirubrum8
11'5; 11·9
10'1° : 1·3 : 4'3 e : 6,9 ; 2·0 : 2'2;
9·8 1·2 4·5 {'1 1·8 2·2
4·4 2·2
-
7·1 13·5 7·4 7·7 14· 7 4·5
-
1l ·7 0·0 10·0 1·3 4·4 7·0 1·9 2·2 0 ·49
2·5 1·3 (1l '2) 4·1 17·2 9·6 7·6 13 '4 4·1 (1l'2) 10·7 0·0 9 ·5 0 ·0 5·2
8·4 2·8 3·6 0·26
9·9 2-1 (7-8) 8· 1 9 ·2 5·8 4·8 11-1 4· 1 (9'0) 12·1 (0'6) 10·6 2·6 6·1 8·2 2·0 3·3 0·99
424
S.T. BAYLEY
acidic proteins during their production, the RNA-rich particles contain a much higher proportion of basic proteins than do whole ribosomes. It is interesting, however, that a significant amount of acidic proteins remains bound to the RNA moiety even at low ionic strengths. (c) Content of magnesium and potas sium ioWJ
E stimates of Mg2+ and K + contents are presented in Table 2. In calculating these, a molecular weight of 3 X 106 was assumed for whole ribosomes (Bayley & Kushner, 1964). The molar concentrations of RNA-rich particles relative to those of whol e ribosomes were assumed to be proportional to the respecti ve phosphorus conc entrations. TABLE
2
Molar ratios for Mg z+ and K+ in ribosomal particles The values are averages for 4 separate preparations of particles Molar ratios
Whole ribosomes RNA·rich particles 1 RNA.rich particles 2t Pea seedling ribosomes (Ts'o, Bonner & Vinograd, 1958) Rabbit r eticulocyte ribosomes (Edelman, et al., 1960)
RNA Protein
Particles
1·4 ± 0·1 3·3 ± 0·3 4'0 ± 0·2
18·4 ± 0·8 X 103 2·9 ± 0'5 x 103 0·6 ± 0'2x 103
M g 2+ Particles
1·7 1·6 1·5
±
0·2
X
Phosphorus
103
0·34
±
0·03
± 0·3 X 103 0·30 ± 0·04 ± 1·0 xl03 0·30 ± 0·18
1' 5 X 103
9·3
0·3
t These are equivalent to the RNA·rich particles on whi ch amino acid analyaes and gel electrophoresis were carried out. Although the K and Mg analyses for RNA-rich particles 2 varied rather widely, Table 2 shows clearly that as the particles were washed with dilute salt solution, the K+ content diminished markedly, while the Mg2+ content remained essentially constant. It is possible that much of the K + found in whole ribosomes was due to contamination from solution D; the effect of such contamination on the Mg2+ content would have been below experimental error, since the concentration ofMg2+ in solution Dis 40 times smaller than that of K +. However, it seems unlikely that contamination was a major source of error, since the volume ofD required to account for the observed lev els of K + would have been unreasonably large-about half the volume of the initial, solid ribosomal pellet, in fact. Although contamination cannot be entirely neglected, it seems reasonable to conclude therefore that whole ribosomes contain appreciable amounts of K + and that this is lost, together with structural proteins, during washing with dilute salt solution. The Mg2+ contents of these particles are very similar to the values, also given in Table 2, for pea seedling ribosomes (Ts'o, Bonner & Vinograd, 1958) and rabbit reticulocyte ribosomes (Edelman ei al., 1960). This point is discussed below.
COMPOSITION OF HALOPHILE RIBOSOMES
425
4. Discussion The present results demonstrate that in addition to the large amount of acidic proteins reported earlier (Bayley & Kushner, 1964), the ribosomes of H. cutirubrum also contain a significant quantity of basic proteins. Most of these and some of the acidic proteins are retained on the ribosomal RNA even at low ionic strengths. The dissolution of the majority of the acidic proteins, which occurs on lowering the ionic strength, is accompanied by a marked drop in the K + content, whereas the Mg2 + content remains roughly constant at a level comparable to that found by Ts'o and co-workers (Ts' 0, Bonner & Vinograd, 1958; Edelman et al., 1960) for ribosomes of nonhalophilic organisms. The conclusion of these authors, that Mg2 + is responsible for stabilizing the configuration of ribosomal RNA but is not critically involved in binding protein to the RNA, therefore appears to apply to halophile ribosomes as well. This is contrary to an earlier suggestion concerning protein binding in halophile ribosomes (Bayley & Kushner, 1964). The unusually high Mg2 + level of 0·1 M required in the external medium by these ribosomes (Bayley & Kushner, 1964) is probably to overcome the effect of excess K + ions on the binding of Mg2 + to the RNA, an effect which Edelman et al. (1960) examined at much lower ionic strengths. In the RNA-rich particles, electrostatic bonds between the basic proteins on one hand and the phosphate groups of the RNA and the acidic proteins on the other, probably stabilize the ribonucleoprotein complex at low ionic strengths. The forces stabilizing the nucleoprotein complex in whole ribosomes at high ionic strengths are less clear, however. It is possible that once the negative charges which predominate on the protein and RNA have been effectively neutralized by potassium ions, hydrogen and hydrophobic bonds are formed. Certainly the bonds involved are sufficiently specific for the acidic proteins to be reabsorbed to the RNA-rich particles to produce particles resembling the original ribosomes (Bayley, manuscript in preparation). Although other species of bacteria evidently contain some acidic as well as basic ribosomal proteins (Waller, 1964), the ribosomes of H. cutirubrum seem unusual in containing appreciable amounts of proteins with isoelectric points of about 3·9 (Bayley & Kushner, 1964) as well as others with isoeleetric points above about 8·5. The main feature that distinguishes the acidic ribosomal proteins of H. cutirubrum from basic ribosomal proteins of this and other organisms (Table 1; also Spahr, 1962) appears to be the relative contents of acidic and basic residues. 'I's'o, Bonner & Dintzis (1958) pointed out that ribosomal proteins are rich in the basic amino acids, arginine and lysine, and the dicarboxylic amino acids, aspartic and glutamic. In Table 1, the total content of these four residues in each of the H. cutirubrum ribosomal samples, as well as in that of E. coli, is close to 40%. The increased content of aspartic and glutamic acid residues in the soluble proteins is therefore compensated by a decrease in the content of arginine and lysine. In view of the rather selective change from basic to acidic residues which is apparent in much of this ribosomal protein compared to non-halophilic ribosomal proteins, and in view of the recent advances made by Nirenberg and co-workers (Nirenberg et al., 1965) in deciphering the RNA code, it is interesting to compare the nucleotide sequences corresponding to acidic and basic amino acids. The relevant triplets are listed in Table 3. It is apparent from these that many of the codons for acidic and basic residues differ only by the base in the 5'-terminal position. Indeed, a significant 30
S. T. BAYLEY
426
TABLE
3
Nucleotide sequences of RNA codons for acidic and basic amino acids from Nirenberg et al, (1965). These authors tested the trinucleotides in bold-face type experimentally as nucleoside diphosphaies and predicted the others Aspartic acid Glutamic acid Asparagine Glutamine Lysine Arginine
GAU
GAC
AAU
AAC
CGU
CGC
GAA
GAG
CAA AAA AGA
CAG AAG AGG
CGA
CGG
change in the acidity of a protein being synthesized might be effected merely by misreading the purines G and A in this position; cf.-Asp-Asn and Glu-Lys. Nirenberg et al. (1965) have suggested that, particularly in codons involving two or more purines, correct recognition of two out of three bases in a trinucleotide may often suffice in protein synthesis. The general similarity of the triplets in Table 3 and the fact that most of them contain two or more purines suggest that this may be one way in which an acidic protein could be synthesized in place of a basic protein. The extreme halophiles appear to be characterized by markedly acidic proteins. It is an interesting question therefore whether extreme halophilism developed through modifications in the recognition of codons similar to those just described, perhaps as a result of high concentrations of cations in the cell. I thank Mr J. L. Labelle for helpful suggestions on the amino acid analyses and for operating the analyser; and Mr C. F. Rollin for expert technical aaaietanoe at all stagea of the work. This paper is issued as National Research Council contribution no. 8849.
REFERENCES Allen, R. J. L. (1940). Biochem, J. 34, 858. Bayley, S. T. & Kushner, D. J. (1964). J. Mol. Biol. 9, 654. Cohn, P. (1962). Biochem. J. 84, 16P. Crampton, C. F. & Petermann, M. L. (1959). J. Biol. Ohem, 234, 2642. Edelman, 1. S., Ts'o, P. O. P . & Vinograd, J. (1960). Biochim. biophya. Acta, 43, 393. Kushner, D. J., Bayley, S. T., Boring, J., Kates, M. & Gibbous, N. E. (1964). Ganad. J. Microbiol. 10, 483. Lowry, O. R., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 11., 265. Marier, J. R. & Boulet, M. (1959) . J. Dairy Sci. 42,981. Mathias, A. P. & Williamson, R. (1964). J. Mol. Biol. 9, 498. Moller, W. J. (1964). Proc, Nat. Acad. s«; Wash. 51, 501. Moore, S. & Stein, W. R. (1963). In Methods in Enzymology, ed, by S. P. Colowick & N. O. Kaplan, vol. 6, p. 819. New York: Academic Press. Nirenberg, M., Leder, P., Bernfield, M., Brimaeombe, R., Trupin, J., Rottman, F. & O'Neal, C. (1965). Proc. Nat. Acad. s«, Wash. 53, 1161. Setterfield, G., Neelin, J. M., Neelin, E. M. & Bayley, S. T. (1960). J. Mol. BioI. J, 416. Spahr, P. F. (1962). J. Mol. BioZ. 4, 395.
COMPOSITION OF HALOPHILE RIBOSOMES
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Swift, H. (1955). In The Nucleic Acids, ed, by E. Chargaff & J. N. Davidson, vol. 2, p. 51. New York: Academic Press. Ts'o, P. O. P., Bonner, J. & Dintzis, H. (1958). Arch. Biochem. Biophys. 76, 225. Ts'o, P. O. P .. Bonner, J. & Vinograd, J. (1958). Biochim, biophys. Acta, 30, 570. Waller, J. P. (1964). J. Mol. Biol. 10, 319. Waller, J. P. & Harris, J. 1. (1961). Proc. Nat. Acad. Sei., Wash. 47, 18. Zacharius, R. M. & Talley, E. A. (1962). Analyt. Ohem, 34, 1551.
Composition of Ribosomes of an Extremely Halophilic Bacterium S. T.
BAYLEY
Dioieum. of Biosciencee National Beseardi Council Ottawa, Ontario, Canada (Received 15 September 1965) Amino acid analyses and electrophoresis on starch-urea gels have shown that the ribosomes of the extremely halophilic bacterium, Halobacterium cutirubrum, contain a majority of acidic proteins with isoelectric points of about 3·9 and a. smaller proportion of basic proteins with isoelectric points above about 8·5. From analyses of magnesium and potassium, it is concluded that magnesium ions probably stabilize the RNA moiety as in other ribosomes, and that potassium ions neutralize negative charges, thereby enabling the acidic proteins to be bound to the ribosomal RNA by hydrogen and hydrophobic bonds. The acidic and basic proteins are similar in amino acid composition except for the relative proportions of acidic to basic residues. The possible significance of this observation in relation to halophilism is discussed.
1. Introduction In ribosomes from a wide variety of sources such as E8cherichia coli (Waller & Harris, 1961; Spahr, 1962; Waller, 1964), pea seedlings (Setterfield, Neelin, Neelin & Bayley, 1960), rat liver (Crampton & Petermann, 1959) and rabbit reticulocytes (Cohn, 1962; Mathias & Williamson, 1964), the structural proteins are predominantly basic and appear to be bound to the phosphate groups of the RNA (Edelman, Ts'o & Vinograd, 1960). An exception to this general pattern was found recently in the extremely halophilic bacterium, Halobacterium cutirubrum (Bayley & Kushner, 1964). The ribosomes from this organism are stable as 70 s particles in 4 M-KCl; 0·1 M-MgCl2 and 0·01 M-tris buffer (pH 7'2). In 0·005 M-tris with 0·005 y-MgCl 2 , roughly two-thirds of the structural protein dissolves to leave RNA-rich particles which sediment at rates similar to those of the RNA moieties of E. coli ribosomes (Bayley & Kushner, 1964; Moller, 1964). Electrophoretic studies showed that the great majority of the dissolved proteins were acidic, with isoelectric points of about 3·9. It is believed that these proteins remain associated with the negatively charged RNA only so long as the negative charges in the complex are shielded by suitable concentrations of cations. To investigate the ribosomal proteins of this halophile further, amino acid analyses have been carried out on whole ribosomes, on the protein which dissolves at low salt concentrations and on the RNA-rich particles which remain. Conclusions reached from these analyses have been confirmed by electrophoresis of the proteins on starch-urea gels. To examine the manner in which these proteins are bound to the ribosomal RNA, the Mg and K contents of ribosomal particles have also been estimated. 420
COMPOSITION OF HALOPHILE RIBOSOMES
421
2. Materials and Methods Ribosomes from H. cutirubrum were prepared in 4 M-KCI; 0·1 M-MgCI:a and 0·01 M-tris buffer (pH 7,2) (solution D), and purified by 2 cycles of differential centrifugation as previously described (Bayley & Kushner, 1964), except that the harvested, washed cells were ground with 1 to 1·5 times their wet weight of alumina, instead of being shaken with ballotini beads. (a) Amino acid analyses Two separate sets of ribosomal protein samples were prepared and analysed. In each experiment, the final ribosomal suspension was centrifuged at 50,000 rev.fmin for 90 min and the ribosomal pellet was resuspended in 0·005 M-tris-MgCI 2 (0,005 M-TMg) (experiment 1) or 0·005 M-tris-MgCI 2-KCI (0,005 M-TMgK) (experiment 2) to a final concentration of about 1 %. A sample of this suspension, representing whole ribosomes, was kept for analysis, while the remainder was centrifuged at 50,000 rev.fmin for 180 min to sediment the RNA-rich particles. The supernatant fraction, containing the soluble protein, was kept for analysis, while the pellet was resuspended in fresh low-salt buffer. The suspension was centrifuged at 50,000 rev.fmin for 180 min and the final pellet, after resuspension in fresh buffer, was analysed as the RNA-rich particles. All samples were freeze-dried. For the amino acid analyses, two portions of each sample, each containing roughly 4 mg protein, were digested after the method of Moore & Stein (1963) for periods of 20 hr and 70 hr, respectively. Because of their high RNA contents, the samples of whole ribosomes and of RNA-rich particles showed extensive humin formation. These were therefore centrifuged or filtered after hydrolysis to remove the deposits. Analyses were carried out on a Beckman Spinco model 120B amino acid analyser. In experiment 1 alone, the analyses were duplicated and agreed closely.
(b) Other chemical analyses Samples to be analysed for Mg and K were prepared as follows. A ribosomal suspension in solution D was centrifuged at 50,000 rev.fmin for 90 min. The supernatant solution was discarded and the ribosomal pellet and the walls of the tube were carefully rinsed twice with 0·005 M-TMgK to remove excess D. The pellet was then resuspended in 0'005 M-TMgK to yield whole ribosomes. This suspension was then centrifuged at 50,000 rev.f min for 180 min to yield a pellet, part of which was resuspended in water and repre· sented RNA-rich particles 1. The remainder was resuspended in 0·005 M-TMgK and centrifuged as before. The resulting pellet was resuspended in water to yield RNA-rich particles 2. Potassium was estimated with a Beckman flame spectrophotometer. To avoid interference from phosphorus and other cations, Mg was estimated from t.ho turbidity formed on adding potassium erucicate, after the method of Marier & Boulet (1959). Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951), using plasma albumin as a standard, and phosphorus by the method of Allen (1940). The weight of RNA was taken as 11 times the weight of phosphorus. (c) Gel electrophoresis Samples of whole ribosomes and of RNA-rich particles were prepared in the same manner as for amino acid analyses, except that the final suspension in each case was in pH 5·2 acetate or pH 8·0 veronal buffer of ionic strength 0·02 at a concentration of about 14 mg protein/nil. Before electrophoresis, 420 mg urea was added to each 1 ml. of sample. To digest the ribosomal RNA so as to release the protein for electrophoresis, a solution of pancreatic RNase (Worthington), pretreated to destroy proteolytic activity (Swift, 1955), was then added, after the method of Waller (1964), to give a concentration of roughly 1 p,g RNasefmg RNA in the sample. Electrophoresis was carried out horizontally in starchurea gels as previously described (Bayley & Kushner, 1964).
422
S.T. BAYLEY
3. Results (a) Amino acid analyses
In the two separate preparations analysed, the ratios of RNA to protein for whole ribosomes were l·ll and 1'36; for soluble protein, 0·051 and 0·1l6; and for RNA-rich particles, 3·4 and 4'09, respectively. In each case, the soluble protein fraction accounted for 60 to 65%, and the RNA-rich particles for about 30%, of the total ribosomal protein. The results of the amino acid analyses for the two preparations are given in Table 1. With the exceptions noted, the values quoted are from the 20-hour hydrolysates. In the analyses of the RNA-rich particles, a measurable peak occurred between alanine and valine, whether the 50°C or the 30 and 50°C programme (Zacharius & Talley, 1962) was used on the column. This material was therefore not cystine, which in the latter programme should be eluted after valine (Zaoharius & Talley, 1962), but it has not been examined further. With whole ribosomes and RNA-rich particles, values have been omitted for ammonia and glycine because of the production of these substances from RNA. For soluble proteins, the ammonia value is taken as an approximate measure of amide nitrogen (Moore & Stein, 1963). The total weight of all the components listed per 100 g soluble protein was estimated to be 70·0 g and 110·7 g in the two experiments, respectively. As a comparison, Table 1 includes a less accurate analysis of the proteins in isolated cell envelopes of H. cutirubrum (Kushner, Bayley, Boring, Kates & Gibbons, 1964), as well as the thorough analysis by Spahr (1962) of the protein from 70 s E. coli ribosomes. In content of neutral residues, there are significant differences between the proteins listed in Table 1, although, for the soluble proteins and the proteins associated with the RNA-rich particles of H. cutirubrum, these differences are small. The distinctive feature of most of the halophile proteins is the high content of acidic, and the relatively low content of basic, amino acids. The excess of aspartic and glutamic acids, less amide ammonia, over lysine, histidine and arginine is 7·6 moles/lOOmoles in the soluble ribosomal proteins, exceeded only by the envelope protein with 11·5. By contrast, the ribosomal proteins of E. coli have an excess of basic over acidic amino acids of 7·6 moles %. (This differs from the figure given by Spahr (1962), because some of the recovered residues have been omitted from the total considered here.) The excess of acidic residues in the soluble ribosomal proteins supports the electrophoretic evidence for the acidic nature of these proteins (Bayley & Kushner, 1964). The ratios of basic to acidic residues (Table 1) suggest that the proteins which remain associated with the RNA in the RNA-rich particles are considerably more basic than those that dissolve. However, it is evident from the results of gel electrophoresis which follow that the analysis for RNA-rich particles actually represents an average of both acidic and basic proteins. (b) Gel electrophoresis
To examine the basicity of the proteins of the RNA-rich particles further, samples of whole ribosomes and of RNA-rich particles were subjected to electrophoresis in starch-urea gels in the presence of RNase (Waller, 1964) as described above. The patterns in Plate I, together with that obtained from whole ribosomes in veronal buffer (not shown), clearly demonstrate that these ribosomes contain proteins with an isoelectric point above about 8·5. As is to be expected from the extensive loss of
(a)
(b)
(c)
PLATE 1. (a) (b) (c) Electrophoretic p atterns in starch-urea gel sta ined with n ig ro s in , aft er 6 hr at i rn A/gel , of (a) whol e H . cutirubrum ribosomes and (b ), (e) R X A-r ieh p articles, all treated overn ight wi th R'Nase in urea . The gels wer e made up (a ) a nd (b ) in aceta te buffer (pH 5,2) a nd (c ) in ver orial b uffer (p H 8 '0 ), a lt ho ug h the pH val ues in the gels were 0·3 t o 0·;; unit h ig her. The anode is at the bottom. R esu lt s wit h amid o black stainin g we re simi la r . On an RNA b a sis, the quantities of RNA-rich part icles in (b) and (c) wer e 1·5 times tha t of whole ribosomes in (a). [facing p . i t :!
TABLE
1
Amino acid analyses, expressed as moles/100 moles amino acids recovered, for (a ) soluble ribosomal protein, (b) RNA-rich particles, and (c) whole ribosomes from H. cutirubrum compared to (d) envelope protein of H . cutirubrum, and (e) 70 s E. coli ribosomal protein Amino ac id
Meana Lysine Histidine Ammonia b • d Arginine Aspartic acid Threonine" Serine> Glutamic acid Proline GlycineAlanine Half-eyst.ino'[ Valine Methionine Isoleucine Leucine Tyrosino'' Phenylalanine Ratio
a b
o d 8
Lys
+ His + Arg + Asp
GIu
3·6 : 2·3 ; (8'{ ; 6·4 ; 14·6;
3'{ 2·0 12' 7) 6·5 14 ·9
{'O ;
{j08
{ 'O :
15 ·1 ; 4·3 ; (9'1 ; 12·0:
4·0 16·5 4·2 10'3 ) 13·0
10.2°: 1·5 : 4'5° ; 7·4 ; 2·2 ; 2·2;
10·6 1·3 5·1° 7·9 2·0 2·3
Whole H. cutirubru m ribosomes
RNA-rich particles
Soluble protein
3·7 2·1 (10'7) 6·4 14·7 6·4 5·5 15·8 4·3 (9'7) 12·5 0·0 10·4 1·4 4·8 7·6 2·1 2·3
6·5 2·2
4·9 ; 4·0 2·2 : 2·1
10·3; 11'6; {'6 ; 7'{ ; 11·8; 5·0 ;
10·6 13 '{ 5·3 3·2 13·6 5·3
10·5 12,7 6 ·5 5·4 12·7 5· 1
13 ·3 ; 12·9
13·1 tr? 9·4 1·3 4·6 6·4 1·4 2·2
{'6 ; 13 ·4 : {'1 : {'8 ; 14,2; 4'6 ;
6·8 13 '{ {'{
7·6 15 ·3 4·5
8·8°; 1·3 : 4'2°: 6·0 ; 2·0 ; 2'1;
9·9° 1·2 5.0 0 6·8 0·8 2·4
0 ·4
Mean of the result s from experimen t s 1 and 2 shown in italics. Extrapolated to zero hydrolysis time (Moore & Stein, 1963 ). Value for 70-hr hydrolysate (Moore & Stein, 1963: Spahr, 1962). Not included in total. From Kushner et al, ( 1964). From Spahr (1962); 0. small amount. of tryptophan was also found.
0·76
70 s E . coli ribosomal protein!
Mean a
Mean a
6·3 : 6·9 2·0 : 2·4
Envelope protein from H. cutirubrum8
11'5; 11·9
10'1° : 1·3 : 4'3 e : 6,9 ; 2·0 : 2'2;
9·8 1·2 4·5 {'1 1·8 2·2
4·4 2·2
-
7·1 13·5 7·4 7·7 14· 7 4·5
-
1l ·7 0·0 10·0 1·3 4·4 7·0 1·9 2·2 0 ·49
2·5 1·3 (1l '2) 4·1 17·2 9·6 7·6 13 '4 4·1 (1l'2) 10·7 0·0 9 ·5 0 ·0 5·2
8·4 2·8 3·6 0·26
9·9 2-1 (7-8) 8· 1 9 ·2 5·8 4·8 11-1 4· 1 (9'0) 12·1 (0'6) 10·6 2·6 6·1 8·2 2·0 3·3 0·99
424
S.T. BAYLEY
acidic proteins during their production, the RNA-rich particles contain a much higher proportion of basic proteins than do whole ribosomes. It is interesting, however, that a significant amount of acidic proteins remains bound to the RNA moiety even at low ionic strengths. (c) Content of magnesium and potas sium ioWJ
E stimates of Mg2+ and K + contents are presented in Table 2. In calculating these, a molecular weight of 3 X 106 was assumed for whole ribosomes (Bayley & Kushner, 1964). The molar concentrations of RNA-rich particles relative to those of whol e ribosomes were assumed to be proportional to the respecti ve phosphorus conc entrations. TABLE
2
Molar ratios for Mg z+ and K+ in ribosomal particles The values are averages for 4 separate preparations of particles Molar ratios
Whole ribosomes RNA·rich particles 1 RNA.rich particles 2t Pea seedling ribosomes (Ts'o, Bonner & Vinograd, 1958) Rabbit r eticulocyte ribosomes (Edelman, et al., 1960)
RNA Protein
Particles
1·4 ± 0·1 3·3 ± 0·3 4'0 ± 0·2
18·4 ± 0·8 X 103 2·9 ± 0'5 x 103 0·6 ± 0'2x 103
M g 2+ Particles
1·7 1·6 1·5
±
0·2
X
Phosphorus
103
0·34
±
0·03
± 0·3 X 103 0·30 ± 0·04 ± 1·0 xl03 0·30 ± 0·18
1' 5 X 103
9·3
0·3
t These are equivalent to the RNA·rich particles on whi ch amino acid analyaes and gel electrophoresis were carried out. Although the K and Mg analyses for RNA-rich particles 2 varied rather widely, Table 2 shows clearly that as the particles were washed with dilute salt solution, the K+ content diminished markedly, while the Mg2+ content remained essentially constant. It is possible that much of the K + found in whole ribosomes was due to contamination from solution D; the effect of such contamination on the Mg2+ content would have been below experimental error, since the concentration ofMg2+ in solution Dis 40 times smaller than that of K +. However, it seems unlikely that contamination was a major source of error, since the volume ofD required to account for the observed lev els of K + would have been unreasonably large-about half the volume of the initial, solid ribosomal pellet, in fact. Although contamination cannot be entirely neglected, it seems reasonable to conclude therefore that whole ribosomes contain appreciable amounts of K + and that this is lost, together with structural proteins, during washing with dilute salt solution. The Mg2+ contents of these particles are very similar to the values, also given in Table 2, for pea seedling ribosomes (Ts'o, Bonner & Vinograd, 1958) and rabbit reticulocyte ribosomes (Edelman ei al., 1960). This point is discussed below.
COMPOSITION OF HALOPHILE RIBOSOMES
425
4. Discussion The present results demonstrate that in addition to the large amount of acidic proteins reported earlier (Bayley & Kushner, 1964), the ribosomes of H. cutirubrum also contain a significant quantity of basic proteins. Most of these and some of the acidic proteins are retained on the ribosomal RNA even at low ionic strengths. The dissolution of the majority of the acidic proteins, which occurs on lowering the ionic strength, is accompanied by a marked drop in the K + content, whereas the Mg2 + content remains roughly constant at a level comparable to that found by Ts'o and co-workers (Ts' 0, Bonner & Vinograd, 1958; Edelman et al., 1960) for ribosomes of nonhalophilic organisms. The conclusion of these authors, that Mg2 + is responsible for stabilizing the configuration of ribosomal RNA but is not critically involved in binding protein to the RNA, therefore appears to apply to halophile ribosomes as well. This is contrary to an earlier suggestion concerning protein binding in halophile ribosomes (Bayley & Kushner, 1964). The unusually high Mg2 + level of 0·1 M required in the external medium by these ribosomes (Bayley & Kushner, 1964) is probably to overcome the effect of excess K + ions on the binding of Mg2 + to the RNA, an effect which Edelman et al. (1960) examined at much lower ionic strengths. In the RNA-rich particles, electrostatic bonds between the basic proteins on one hand and the phosphate groups of the RNA and the acidic proteins on the other, probably stabilize the ribonucleoprotein complex at low ionic strengths. The forces stabilizing the nucleoprotein complex in whole ribosomes at high ionic strengths are less clear, however. It is possible that once the negative charges which predominate on the protein and RNA have been effectively neutralized by potassium ions, hydrogen and hydrophobic bonds are formed. Certainly the bonds involved are sufficiently specific for the acidic proteins to be reabsorbed to the RNA-rich particles to produce particles resembling the original ribosomes (Bayley, manuscript in preparation). Although other species of bacteria evidently contain some acidic as well as basic ribosomal proteins (Waller, 1964), the ribosomes of H. cutirubrum seem unusual in containing appreciable amounts of proteins with isoelectric points of about 3·9 (Bayley & Kushner, 1964) as well as others with isoeleetric points above about 8·5. The main feature that distinguishes the acidic ribosomal proteins of H. cutirubrum from basic ribosomal proteins of this and other organisms (Table 1; also Spahr, 1962) appears to be the relative contents of acidic and basic residues. 'I's'o, Bonner & Dintzis (1958) pointed out that ribosomal proteins are rich in the basic amino acids, arginine and lysine, and the dicarboxylic amino acids, aspartic and glutamic. In Table 1, the total content of these four residues in each of the H. cutirubrum ribosomal samples, as well as in that of E. coli, is close to 40%. The increased content of aspartic and glutamic acid residues in the soluble proteins is therefore compensated by a decrease in the content of arginine and lysine. In view of the rather selective change from basic to acidic residues which is apparent in much of this ribosomal protein compared to non-halophilic ribosomal proteins, and in view of the recent advances made by Nirenberg and co-workers (Nirenberg et al., 1965) in deciphering the RNA code, it is interesting to compare the nucleotide sequences corresponding to acidic and basic amino acids. The relevant triplets are listed in Table 3. It is apparent from these that many of the codons for acidic and basic residues differ only by the base in the 5'-terminal position. Indeed, a significant 30
S. T. BAYLEY
426
TABLE
3
Nucleotide sequences of RNA codons for acidic and basic amino acids from Nirenberg et al, (1965). These authors tested the trinucleotides in bold-face type experimentally as nucleoside diphosphaies and predicted the others Aspartic acid Glutamic acid Asparagine Glutamine Lysine Arginine
GAU
GAC
AAU
AAC
CGU
CGC
GAA
GAG
CAA AAA AGA
CAG AAG AGG
CGA
CGG
change in the acidity of a protein being synthesized might be effected merely by misreading the purines G and A in this position; cf.-Asp-Asn and Glu-Lys. Nirenberg et al. (1965) have suggested that, particularly in codons involving two or more purines, correct recognition of two out of three bases in a trinucleotide may often suffice in protein synthesis. The general similarity of the triplets in Table 3 and the fact that most of them contain two or more purines suggest that this may be one way in which an acidic protein could be synthesized in place of a basic protein. The extreme halophiles appear to be characterized by markedly acidic proteins. It is an interesting question therefore whether extreme halophilism developed through modifications in the recognition of codons similar to those just described, perhaps as a result of high concentrations of cations in the cell. I thank Mr J. L. Labelle for helpful suggestions on the amino acid analyses and for operating the analyser; and Mr C. F. Rollin for expert technical aaaietanoe at all stagea of the work. This paper is issued as National Research Council contribution no. 8849.
REFERENCES Allen, R. J. L. (1940). Biochem, J. 34, 858. Bayley, S. T. & Kushner, D. J. (1964). J. Mol. Biol. 9, 654. Cohn, P. (1962). Biochem. J. 84, 16P. Crampton, C. F. & Petermann, M. L. (1959). J. Biol. Ohem, 234, 2642. Edelman, 1. S., Ts'o, P. O. P . & Vinograd, J. (1960). Biochim. biophya. Acta, 43, 393. Kushner, D. J., Bayley, S. T., Boring, J., Kates, M. & Gibbous, N. E. (1964). Ganad. J. Microbiol. 10, 483. Lowry, O. R., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. BioZ. Chem. 11., 265. Marier, J. R. & Boulet, M. (1959) . J. Dairy Sci. 42,981. Mathias, A. P. & Williamson, R. (1964). J. Mol. Biol. 9, 498. Moller, W. J. (1964). Proc, Nat. Acad. s«; Wash. 51, 501. Moore, S. & Stein, W. R. (1963). In Methods in Enzymology, ed, by S. P. Colowick & N. O. Kaplan, vol. 6, p. 819. New York: Academic Press. Nirenberg, M., Leder, P., Bernfield, M., Brimaeombe, R., Trupin, J., Rottman, F. & O'Neal, C. (1965). Proc. Nat. Acad. s«, Wash. 53, 1161. Setterfield, G., Neelin, J. M., Neelin, E. M. & Bayley, S. T. (1960). J. Mol. BioI. J, 416. Spahr, P. F. (1962). J. Mol. BioZ. 4, 395.
COMPOSITION OF HALOPHILE RIBOSOMES
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Swift, H. (1955). In The Nucleic Acids, ed, by E. Chargaff & J. N. Davidson, vol. 2, p. 51. New York: Academic Press. Ts'o, P. O. P., Bonner, J. & Dintzis, H. (1958). Arch. Biochem. Biophys. 76, 225. Ts'o, P. O. P .. Bonner, J. & Vinograd, J. (1958). Biochim, biophys. Acta, 30, 570. Waller, J. P. (1964). J. Mol. Biol. 10, 319. Waller, J. P. & Harris, J. 1. (1961). Proc. Nat. Acad. Sei., Wash. 47, 18. Zacharius, R. M. & Talley, E. A. (1962). Analyt. Ohem, 34, 1551.