Fractionation of Escherichia coli S-RNA on dextran gels (Sephadex)

J. Mol. Biol. (1965) 11,458-466

Fractionation of Escherichia coli S-RNA on Dextran Gels (Sephadex) R. ROSCHENTHALER AND

P.

FROMAGEOT

a

Service de Biochimie du Departement de Biologie, Oommissariat l'Energie Atomique O.E.N. Saclay, Gif-Bur-Yvette, S. et 0., France (Received 21 September 1964) A method for fractionation of S-RNA by gel filtration is described. It has been shown that the individual S-RNA's are distributed into two groups and that they are partially fractionated within these groups. The possible reasons for the type of fractionation are discussed.

1. Introduction The interest in a fractionation of S·RNA has already been admirably discussed (Crick, 1958,1963), and it need not be further emphasized here. The methods attempted are various (Schweet, Bovard, Allen & Glassman, 1958; Cantoni, 1960; Lipshitz & Chargaff, 1960; Hartmann & Coy, 1961; Stephenson & Zamecnik, 1962; Zubay, 1962; Jacobson & Nishimura, 1963; Sueoka & Yamane, 1962). Counter-current distribution (Holley & Merrill, 1959; Apgar, Holley & Merrill, 1962; Tada, Schweiger & Zachau, 1962; Tanaka, Richards & Cantoni, 1962; Goldstein, Bennett & Craig, 1964) especially has resulted in preparative separation, and it seems to be the most promising method today. However, the counter-current distribution procedure is cumbersome and therefore we sought a simpler method to obtain at least a fractionation into groups of S-RNA on a milligram scale, by using filtration on dextran gel columns (Sephadex). In principle, the method is feasible, since it is known that dextran gels are able to separate molecules differing in size; for example, it has been used to separate ribosomal RNA and S-RNA (Dirheimer & Ebel, 1964). As the physical properties of S-RNA, for example hypochromicity, are dependent upon ionic strength, especially in the presence of Mg2 + , it was hoped that conditions might be found under which the molecular configurations of the various S-RNA species might be sufficiently altered to allow separation by gel filtration. The present results demonstrate that Escherichia coli S-RNA can indeed be fractionated on Sephadex columns.

2. Materials and Methods S·RNA The S-RNA (sodium salt) was supplied by General Biochemicals, Chagrin Falls (Ohio) and was isolated from E. coli, strain B. This material contains less than 0'5% DNA as checked by the diphenylamine test. The protein content is not measurable with the Folin-Ciocalteau reagent using the method of Lowry, Rosebrough, Farr & Randall (1951). With ninhydrin, no reaction is detectable. The S·RNA used is stripped from esterified amino acids. A water solution of 1 mgjml. of this S-RNA has an optical density of 24 at 260 mp.. On centrifugation (0,5 mg) in a sucrose density-gradient (5 to 20%), 458

FRACTIONATION OF ESCHERICHIA COLI S-RNA

45'9

the preparation as purchased gives one broad peak, as described by Nihei & Cantoni (1963) for example. Mter dialysing (2 hr against water), the material had the following acceptor activities, in mp.molesjmg, calculated on the basis of O.D. measurement at 260 mJL: valine, 4·3; glycine, 3·35; arginine, 4'4; proline, 0·845; histidine, 2'14; lysine, 2'44; phenylalanine, 2·43; aspartic acid, 2·31; leucine, 1'51; glutamic acid, 2'34; tyrosine, 1'9; serine, 1'7. Before using, the S·RNA was dialysed against water and further purified by running through a small column of Sephadex G25. If necessary, the outcoming solution of S.RNA was concentrated by lyophilization. The volume of S·RNA solution placed on the separating columns was 0·5 ml.

S ephadez columns Two types of Sephadex were used: GI00 and G75. The columns were packed according to Flodin (1962) and were operated at 4°C. The values "Vo" of some columns (G75) were determined with haemoglobin; the method is not completely satisfactory, as haemoglobin penetrates slightly into the gel particles. Buffer 80lutions The Sephadex columns were equilibrated with a 0'05 M.potassium acetate buffer (pH 5,0), and in one case with 0·35 M-NaCl solution. Mter introduction of the S·RNA sample, the columns were either developed with the solutions described above, or with a linear decreasing NaCl gradient (dilution of 500 ml. of 0·35 M-NaCI solution with 500 ml, of 0'005 M-magnesium acetate in 0'025 M-acetic acid). The flow rate was 8 to 10 ml.jhr, Detection of the specific S. RNA In the 2-rol. fractions eluted from the columns, detection and measurement ofthe amount of acc eptor activity were by the method described by Zubay (1962), on 100·JLl. portions. The HC-labelled amino a cids were prepared by Dr H. Maier-Huser of this laboratory. The specific activities were: alanine, 84·0 mc/m-mole: arginine, 70'0 mo/m-mole: aspartic acid, 47'Ome/m-mole; glutamic acid, 77'Ome/m-mole; glycine, 21'Omo/m-mole; histidine, 48 mc/m-rnole; leucine, 71'0 mo/m-mole: lysine, 74·Omc/m.mole; phenylalanine, 80·0 me/ m-mole; proline, 157·0 m c /m-mole, tyrosine, 53·0 mc/m-mole; serine, 43·0 mc/m-mole; valine, 50·0 mojm-mole, The cysteine was labelled with 35S; its specific activity was about 20 mo/m-mole. The HC-Iabelled hydrolysate was prepared from algae. The supernatant fraction (105,OOO g) from E. coli homogenates used as a source of activating enzymes was filtered on Sephadex G25 to remove free amino acids. The reproducibility of the fixation of an amino acid was found to be within ± 5'7% for charges of 11 equal samples ofS-RNA with leucine; for the other amino acids the errors were smaller.

3. Results Figure 1 shows the profile of the acceptor activities for different amino acids, after a run of 130 mg of uncharged S·RNA through a Gl00 Sephadex column, eluted with NaCl-acetic acid gradient. These profiles have a distinct shape, and their maxima furthermore correspond in nearly all cases with different elution volumes. The acceptor activities are distributed roughly into two groups corresponding with the second and the third peaks of absorbancy of 260 ta«, One group consists of the S·RNA's for glutamic acid, leucine, tyrosine, glycine and serine. The other group corresponds to the S·RNA's for phenylalanine, lysine, proline, aspartic acid, alanine, cysteine and valine. Hi stidine and proline showed two peaks for acceptor activity. Whereas the two histidine peaks were found in both groups, the two proline peaks were found only in the second group . It is also noteworthy that nearly all the specific acceptor activities have an unsymmetrical distribution. If the Gl00 Sephadex column is developed with 0·05 M-potassium acetate (pH 5), instead of the gradient,

460

R. ROSCHENTHALER AND P. FROMAGEOT

the separation of the acceptor activities (100 mg of S-RNA added on the column) is poorer and the two peaks corresponding to the optical density at 260 mf£ are closer together. With a solution 0·05 N of acetic acid alone, the separation of different acceptor activities (tested: leucine, tyrosine, glutamic acid, serine, alanine, cysteine) is still worse. With this solvent only one peak of absorbancy at 260 mf£ is observed. The fractionation of S-RNA can also be observed on G75 Sephadex columns. The three different conditions used are summarized in Table 1. On such columns, the

Serine

Glutamic acid

4 3 2

10

Histidine

Leucine

4

8

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3

6

2 4

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Phenyl-alan ine

Glycine

Proline

II>

v

8 6

4 2

10

4

8

3

6

2

4

89

20289 Tube no.

145

FRACTIONATION OF ESOHERIOHIA OOLI S -RNA

Alanine

Cyste ine

.,

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4

g •, Arginine

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89 FIG.!. Gel filt rat ion of 130 mg S·RNA through a GIOOSephadex column, 160 em X 2·5 em, with 0·35 M-NaCl-0- 005 M-magnesium acetate in 0-025 M-acetic acid-gradient. The solid lines are opt ical density at 260 mp.; the dashed lines radioactivity after charging 100 p.1. of the sample with a HC-Iabelled amino ac id.

elut ion volumes of the different S-RNA's, as detected by their acceptor activities, were measured and the distribution coefficients (K D ) of these activities calculated by reference to the elution volume of haemoglobin. Therefore, the S-RNA eluted at the same volume as haemoglobin has a K D = O. These K D values are referred t o, therefore, as K DH b • It should be noted that since haemoglobin is not completely excluded by the G75 Sephadex particles, the elution volume for haemoglobin is larger than the void volume. Table 1 shows that, for the three conditions used, the 32

462

R. ROSCHENTHALER AND P. FROMAGEOT TABLE

1

Charaderietice of the separation columns Column no.

V.

"Vo"

vlt

(cm'')

(em")

(em")

Length and diameter (em)

Gradient NaCI-acetic acid

555

210

314

109/2'5

0·05 M· potassium acetato (pH 5,0)

0·05M· potassium acetate (pH 5,0)

537

206

298

109/2-;'

0·35 M· N aCI solution

0·35M· N aCI solution

537

199

307

108/2-5

Equilibrated with

Solvent

1

O·05Mpotassium acetate (pH 5,0)

2

3

t VI was calculated by the formula VI = (V. - Vol

W·d (1

where VI is imbibed water in the gel particles "V o" is elution of haemoglobin V. is total bed volume W r is water regain (for G75 = 7'5g/g) d is wet density of the gel (for 0751·03 g/mL)

+r Wr )

elution volumes for haemoglobin are the same, despite the difference in pH from 5·0 (columns 1 and 2) to neutrality (column 3). Therefore haemoglobin behaves as an undissociated molecule in these systems. Figure 2(a), (b) and (c) shows the elution curve of absorbing material at 260 mJL plotted versus K D H b values, and the distribution of the acceptor activities for glutamic acid and valine. The activities are fairly well separated; the former migrates like haemoglobin along columns equilibrated with a potassium acetate buffer of pH 5,0, and is eluted either with the NaCI gradient or with buffer alone. On the contrary, valine acceptor activity is more retained by these two columns. If the Sephadex behaves towards these substances as a molecular sieve, one is led to conclude that, under the conditions used, the acceptor activity for glutamic acid belongs to a molecule the size of which is close to that of haemoglobin, whereas the valine S-RNA would correspond to a molecule of smaller size. Figure 2(c), which refers to a G75 Sephadex column saturated with 0·35 M-NaCI solution, shows also a good separation between glutamic acid and valine acceptor activities, but their positions with respect to haemoglobin are different. The molecule carrying the glutamic acid acceptor activity should be larger than haemoglobin, if molecular sieving is the effective factor for separation. Furthermore, in this column a large amount of material absorbing at 260 mJL appears before haemoglobin (K D H b = - 0,25). This material can scarcely be charged with amino acids, while the total glutamic acid acceptor activity is smaller than in the two preceding systems. Nevertheless, if the material with a K D H b of - 0·25 is heated or dialysed, the acceptor activities increase, as shown in Table 2. This behaviour seems to be quite general although it is less important in other S·RNA fractionation conditions. For example,

FRACTIONATION OF ESCHERI OHIA CO L I S -RNA

463

,-- -,- -,8

2

(0)

6 4 2 00

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(b)

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52

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FIG. 2. Comparative gel filtration on G75 Sephadex. The solid lines are optical density at 260 mJL; the dashed lines are radioactivity after charging with glutamic acid (open circ les) and valine

(t riangles) using as solvent: (a ) a linear gradient of 0·35 M-NaCI-0'005 M.magnesium acet ate in 0·025 M.acetic acid; (b) 0·05 M-potassium acetate buffer; and (c) 0·35 M·NaCI on a column, equilibrated with the same solution. The dashed vertical line marks the elution volume for haemoglobin.

if one runs 146 mg S·RNA under the conditions of Fig. 2(b) (0,05 sr-potaseium acetate, pH 5), and measures in each 5-ml. fraction collected from the column the O.D' 260 and the acceptor activity for arginine, then dialyses each fraction for five hours against water and repeats the measurements, it is found that the O.D ' 260 decreases by 12% and that the acceptor activity increases by 11%. If one assumes that the decrease in O.D. is due to losses by dialysis, one can compute the acceptor activity corresponding to the O.D. before dialysi s. The increase of the acceptor activity due to dialysis is th en found to be abou t 40% for all fractions collected.

464

R. ROSeHENTHALER AND P. FROMAGEOT TABLE

2

Effect of dialysing and heating of the peak material from Fig. 2(c) with a K DH b of - 0·25 Amino acid used for charging

Treatment

Leucine

Dialysedj Heatedt None

Valine

Mean cts/min from 3 experiments

ets/min

% of increase

O.D. 260m"

ofcts/min

429 217 153

26·8 12·6 10·2

280 132 100

Dialysed Heated None

155 212 82

9·7 14·9 5·5

190 274 100

Dialysed Heated None

80 43 22

5·0 2·9 1·5

344 260 100

t 3 ml. of material with the K D H b value about - 0·25 was dialysed against 400 ml, of a solution of 360 ml. water and 40 ml. 0·05 M.potassium acetate, for 3 hr. The measurement of the acceptor activity was calculated for material with the same absorbancy at 260 mIL. t Undialysed material was heated to 95°e and cooled slowly to 37°e in 2 hr. A similar picture is given by the fractions emerging from a G100 Sephadex column developed with 0·05 N-acetic acid. Using identical reagents for testing the leucine acceptor activity in fractions collected and those frozen and stored, a progressive increase of acceptor activity was observed when the fractions were re-assayed four times in five days. The treatments to which these fractions were submitted were only freezing and thawing for the assays. The increase in activity was in the range 150 to 400%. The tyrosine acceptor activity measured in the same fraction remained unaltered. The extent of the recovery in the collected fractions of the material placed on the column has been measured in experiments run under the conditions of Fig. 2(b) (0,05 M-potassium acetate, pH 5). The recovered O.D' 2 6 0 amounts to 88% of the 0.D' 2 6 0 of the material submitted to fractionation. Meanwhile, a loss of 40% of the acceptor activity, as measured with arginine, is observed. If now, the material recovered is lyophilized without removing the salt and re-run on the same column, a loss of 40% of O.D' 2 6 0 is found as well as a loss of 24% acceptor activity, as measured with arginine. In no case can ultraviolet absorbing compounds be washed out of the columns.

4. Discussion The results reported here demonstrate that the mere filtration of E. coli S-RNA through a Sephadex column leads to a fractionation of the different acceptor activities. This fractionation is poor in the absence of salt, and also in the presence of 1 M-NaCI. With G100 Sephadex, the best results were obtained by using a decreasing gradient of 0·35 M-NaCI-0'025 x-acetio acid--O·005 M-magnesium acetate immediately after the S-RNA sample. In this case two main peaks of acceptor activity were found and the corresponding S-RNA's appeared to be separated into two groups. The S-RNA's belonging to these groups are more difficult to separate from each other by Sephadex

FRACTIONATION OF ESOHERIOHIA OOLI S·RNA

465

filtration only, but as their relative position is reproducible, one obtains a pattern useful for other purposes. Most of the profiles for the individual S·RNA's are not symmetrical and some show two peaks (histidine, proline) or shoulders (Fig. 1). Such heterogeneity among S·RNA's has already been described, for example, by Sueoka & Yamane (1962) for leucine, isoleucine, tryptophan and serine, after separation of E. coli S-RNA on methylated albumin columns; heterogeneity has also been demonstrated by Berg, Bergmann, Ofengand & Dieckmann (1961) for methionine, and by Doctor, Apgar & Holley (1961) for leucine and threonine S-RNA's. That this heterogeneity corresponds to a degeneracy of the code has been directly demonstrated by Weisblum, Benzer & Holley (1962) for leucine S.RNA, using polynucleotides as messenger RNA. However, indirect evidence for a degeneracy of the code has been found by other methods (for example, Berg, Lagerkvist & Dieckmann, 1962). There remains the possibility, however, that heterogeneity might be observed for a given S·RNA without there being differences in the anticodons. The fact that on G75 Sephadex, according to the solvent used for developing the column, one can obtain different profiles for the glutamic acid acceptor activity, as well as for the valine acceptor activity, may indicate some kind of association between the S·RNA molecules. This suggestion is in agreement with the observation that glutamic acid S·RNA is eluted from G75 Sephadex at pH 5 with the same elution volume as haemoglobin, and in the presence of neutral NaCl, even before haemoglobin; while another glutamic acceptor activity appears with the same elution volume as valine S·RNA. Furthermore, in the conditions described for Fig. 2(c), a material absorbing at 260 mfL was eluted with a solvent volume corresponding to compounds excluded from the gel. This material can be made to accept more amino acid by heating or by dialysis, as if there were liberation of some active S-RNA originally present in an unreactive form. Also, the increase of acceptor activity for leucine observed in fractions eluted from a Sephadex column, as reported above, leads to similar conclusions, the only difference in the treatment to which these S-RNA fractions were submitted being freezing and thawing. Such observations seem to be similar to those made by Nishimura & Novelli (1964), after treatment of E. coli S-RNA with Bacillus subtilis RNase. Following this line of thought and assuming that the separation on Sephadex relies upon its molecular sieve properties, one is led to suggest that the first group of the separated S-RNA corresponds to some associated molecules, which are eluted with the same elution volume as haemoglobin, whereas the second group corresponds to unassociated material. That E. coli S·RNA, after heating and rapid cooling, can behave in the ultracentrifuge as material having an average molecular weight of 57,000 ± 2000 has been shown by Brown & Zubay (1960). Our treatment of S-RNA with salt after dialysing against water may have an effect on the hydrogen bonds similar to that of heating and rapid cooling. Another possibility is that the shape alone of the S-RNA molecules is responsible for such behaviour, the analogy between K D for glutamic acid S-RNA and haemoglobin being fortuitous and having no bearing on the molecular weight itself but on the secondary or tertiary structure. In this case, the first group of S·RNA's should correspond to molecules of more extended shape than the second one. The choice between these two possibilities is under investigation. The increases shown in Fig. 2(c) in acceptor activities noticed after dialysis or heating the RNA fractions also deserve mention. Whether this increase is correlated

466

R. ROSCHENTHALER AND P. FROMAGEOT

with changes in association between different molecules, or changes in the folding of single species, is not known. However, it is clear that for a given amount of material its acceptor activity depends on the conditions that prevailed before the assay is made. As such pre-treatments are liable to alter also the O.D. 26 0 /P values, the ratio between O.D' 26 0 and acceptor activity of & given sample of S-RNA can be varied. These considerations are in agreement with the apparent discrepancy in the data on recovery of O.D' 260 and acceptor activity from the columns. As this work was completed, a paper by Schleich & Goldstein (1964) appeared, describing the behaviour on Sephadex gels of S-RNA previously purified by countercurrent distribution. The authors observed several O.D' 26 0 peaks corresponding to different molecular species and put forward the notion of S-RNA aggregation, some inactive material being convertible to biologically active material by urea treatment. These results are in good agreement with our findings. We are grateful to Miss Agnes La Roche for technical assistance. REFERENCES Apgar, J., Holley, R. W. & Merrill, S. H. (1962). J. Biol. Ohern, 237, 796. Berg, P., Bergmann, F. H., Ofengand, E. G. & Dieckmann, M. (1961). J. Biol. Chem. 236, 1726. Berg, P., Lagerkvist, U. & Dieckmann. M. (1962). J. Mol. Biol. 5, 159. Brown, G. L. & Zubay, G. (1960). J. Mol. Biol. 2, 287. Cantoni, G. L. (1960). Nature, 188, 300. Crick, F. H. C. (1958). Symp. Soc. Exp. Biol. 12, 138. Crick, F. H. C. (1963). Progr. Nucleic Acid Res. vol. I, 164. Dirheimer, G. & Ebel, J. P. (1964). Bull. Soc. Chim, Biol. 46, 399. Doctor, B. P., Apgar, J. & Holley, R. W. (1961). J. Biol. Chem. 236, 1117. Flodin, P. (1962). Dextran Gels and their Applications. Uppsala: Pharmacia, Goldstein, J., Bennett, T. B. & Craig, L. E. (1964). Proc, Nat. Acad. s«, Wash. 51; 119. Hartmann, G. & Coy, U. (1961). Biochim, biophys. Acta, 47, 612. Holley, R. W. & Merrill, S. H. (1959). J. Amer. Ohern: Soc. 81, 753. Jacobson, K. B. & Nishimura, S. (1963). Biochim. biophys. Acta, 68, 490. Lipshitz, R. & Chargaff, E. (1960). Biochim, biophys. Acta, 42, 544. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. (1951).J. Biol. Chem. 193, 265. Nihei, T. & Cantoni, G. L. (1963). J. Biol. Ohern: 238, 3991. Nishimura, S. & Novelli, G. D. (1964). Biochim. biophys. Acta, 80, 574. Schleich, T. & Goldstein, J. (1964). Proc, Nat. Acad. Sci., Wash. 52, 744. Schweet, R., Bovard, F., Allen, E. & Glassman, E. (1958). Proc, Nat. Acad. Sei., Wash. 44, 173. Stephenson, M. L. & Zamecnik, P. C. (1962). Biochem, Biophys. Res. Comm, 7, 91. Sueoka, N. & Yamane, T. (1962). Proc, Nat. Acad. s«; Wash. 48, 1454. Tada, M., Schweiger, M. & Zachau, H. G. (1962). Hoppe Seyl. Z. 328, 85. Tanaka, K., Richards, H. H. & Cantoni, G. L. (1962). Biochim. biophys. Acta, 61, 846. Weisblum, B., Benzer, S. & Holley, R. W. (1962). Proc, Nat. Acad. Sci., Wash. 48, 1449. Zubay, C. (1962). J. Mol. Biol. 4, 347.