The preparation of transfer ribonucleic acid from Escherichia coli

76

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95834

T H E P R E P A R A T I O N OF T R A N S F E R RIBONUCLEIC ACID FROM E S C H E R I C H I A COLI S I D N E Y GUTCHO

Schwarz BioResearch, Inc., Orangeburg, N . Y . (U.S.A.) (Received August 2ist, 1967) (Revised manuscript received November 3rd, 1967)

SUMMARY

A method for the preparation of tRNA from Escherichia coli is described. A crude tRNA is prepared b y phenol and water treatment of cells without the need of centrifugation for the separation of an aqueous phase from a phenolic phase containing cellular debris. Dimethyl sulfoxide and NaC1 are used for the purification of the tRNA in good yield without a requirement for colulIm chromatography. Amino acid acceptance assays are used to quantitate the purification and to characterize the preparations.

INTRODUCTION

The isolation of transfer ribonucleic acid (tRNA) from microorganisms, particularly from baker's yeast and Escherichia coli, has been described in the biochemical literature by many investigators 1. With the exception of the hot detergent extraction prodecure of OFENGAND, DIECKMANN AND BERG 2 and the solvent extraction procedure of RAMMLER, OKABAYASPIIAND DELK ~, the phenol extraction technique of KIRBY4 has been generally used for recovery of tRNA from yeasts and bacteria. The phenol procedure initially described by MONIER, STEPHENSON AND ZAMECNIK5 was extended by HOLLEY~ to the large-scale preparation of tRNA from baker's yeast. Significant advantages of HOLLEY'S procedure are the syphoning off of an aqueous phase, containing the yeast tRNA, to achieve separation from the phenol and cellular debris and the purification of a crude tRNA by DEAE-cellulose chromatography. In experiments with E. coli, an effective disruption of cells was obtained by treatment with phenol and water in the proportions given by HOLLEY for yeast cells. Recovery of an aqueous phase could not be achieved to give good yields of tRNA without high-speed centrifugation, the conventional manner for the separation of the aqueous phase from the phenol and bacterial debris. However, it was found Abbreviation: tRNA, transfer RNA.

Biochim. Biophys. dcta, 157 (1968) 76-82

E. coli t R N A

77

that the addition of larger amounts of these reagents to E. coli effected a rapid separation of the phases and permitted the syphoning off of the aqueous phase. The resulting disruption of the cells was so thorough that higher molecular weight RNA, DNA and other cellular matter, non-ultraviolet absorbing in nature, were also extracted. This result was thus in contrast to the sieving effect described for the phenol treatment of yeast 5 and for the solvent extraction of t R N A from E. coli 3. To purify effectively and rapidly the initially crude tRNA, a solvent fractionation procedure was developed. Dimethyl sulfoxide under the conditions described in this paper was utilized for this purification. The method resulted in the preparation of t R N A in good yield without a requirement for column chromatography. MATERIALS

E. cogi B was purchased from Grain Processing, Muscatine, Iowa. Liquefied phenol containing no preservative was a product of Mallinckrodt Chem. Works. Dimethyl sulfoxide (DMSO) was a product of Crown Zellerbach, Camas, Washington. Dimethylformamide and dimethylacetamide were products of Du Pont. Sephadex was obtained from Pharmacia Fine Chemicals, Piscataway, N. J. The L-E14Clamino acids were prepared and accurately standardized in this laboratory for assay of tRNA. Solutions with specific activities of 50 mC/mmole contained I m#mole ± 5 % per/~1. Radiochemical purity was greater than 99 %METHODS

Isolafion and purification of E. coli tRNA Preparation o/crude tRNA (Step z). To 227 g of frozen cells were added 425 ml of 88 % phenol. After 1.5-2.o h of stirring at room temperature, the uniform, welldispersed slurry was treated with 99 ° ml of deionized water. After I h of additional stirring, the suspension was left to settle at room temperature. On the following day, an additional 475 ml of 88 °/o phenol were added. The mixture was stirred for o.5 h and then I I i O ml of water were added. Stirring was continued for I h, and the suspension left to settle overnight at room temperature. The top aqueous layer, approx. 2 1, was syphoned off while an additional IOO ml were recovered b y centrifugation of the water-phenol interface at 2500 rev./min. To the aqueous layer, which was slightly cloudy, o.I vol. of 20 % potassium acetate (pH 5.2) and 2 vol. of ethanol were added with agitation. The precipitate containing t R N A settled out overnight, and the clear alcoholic supernatant was syphoned off and discarded. The precipitate was recovered b y centrifugation at low speed, washed once with cold 75 % ethanol and once with cold isopropanol, and dried in vacuo at 25-30 °. The yield of crude t R N A was 5.42 g. Its absorbance in water was 72.0" lO 3 A260 m # units*. First dimethyl sul/oxide purification (Step 2). The crude t R N A was taken up in 0.05 M potassium acetate to give a concentration of 36" lO 3 Aze0 m# units per IOO ml. t A n A , e 0 mv u n i t is e q u i v a l e n t t o a n a b s o r b a n c e o f i a t 2 6 0 m / , g i v e n b y m a t e r i a l d i s s o l v e d in w a t e r a n d m e a s u r e d w i t h a l i g h t p a t h o f I c m .

Biochim. Biophys. Acta, 157 ( 1 9 6 8 ) 7 6 - 8 2

78

S. (;UTCtt~

The pH was raised to 8.8-9.0 with 5 M NH4OH. The suspension was incubated at 37 ° for I h to strip bound amino acids and brought to pH 7.0-7.2 at 15-2o ° with 5 M acetic acid. Dimethyl sulfoxide, 0.70 vol., was slowly added with agitation and chilling so that the temperature of the suspension did not exceed 35 °. At 30-32o , 3 M NaC1, 0.25 of the initial aqueous volume, was added. The suspension was maintained at 30-320 for 0. 5 h prior to centrifugation at 2500-3000 rev./min. The residue was washed with a cold mixture of water-dimethyl sulfoxide-3 M NaC1 (I: 1:0.25, by vol.), 0.25-0.33 of the initial aqueous volume. To the combined, clear supernatants, 0.5 vol. of ethanol was added at 5-1o °. The precipitate was recovered by centrifugation, washed with cold 75 % ethanol and then with cold isopropanol, and dried in vacuo at 25-3 O°. This t R N A fraction weighed 1.722 g. Its absorbance was 31. 4. lO3 A2e0 ms units.

Second dimethyl sul/oxide purification (Step 3). The t R N A fraction was dissolved in water to give a concn, equivalent to 64.1o 8 A260ms units/Ioo ml. This fraction dissolved readily to form a greenish, slightly cloudy solution. An equal volume of dimethyl sulfoxide was added slowly with stirring and chilling so that the temperature did not exceed 30 °. Then, 3 M NaC1 equal to 0.20 of the inital aqueous volume was added, and the suspension cooled to 20-23 °. After 15 min, the suspension was centrifuged and the residue washed as above with a cold mixture of water-dimethyl sulfoxide-3 M NaC1 (I: I :0.2, b y vol.). To the combined, clear supernatants, 0.33 vol. of ethanol was added at 5-1o °. After i h, the product was recovered by centrifugation and washed with cold 75 % ethanol and isopropanol to remove C1-. The t R N A was dried in vacuo at 25-3 o° and stored cold in a desiccator. The yield of t R N A was o.84I g containing 16.6. lO3 A260m/~ units, 19. 7 A260rot, units/mg.

Preparation o / E . coli activating enzyme The procedure described for yeast cells by STEPI-IENSON AND ZAMECNIK7 as modified by HOSKINSON AND KHORANAs was used. The protein fraction prepared from IOO g of E. coli was passed through a 5.8 cm × 23 cm column of Sephadex G-25. The enzyme was stored frozen in small aliquots. Prior to the assay of tRNA, 1. 7 ml containing 34 mg of protein were passed through a 2.8 cm × 28 cm column of Sephadex. Equilibrations and elutions were with 0.05 M Tris sulfate (pH 7.5) containing o.ooi M EDTA. Pooled fractions contained more than I mg of protein/ml and had an A2s0 m~/ A2e0 ms of about 1.5.

Assay o/amino acid acceptance activity Activity of the t R N A was determined by a procedure similar to that described by BERG et al. °. The incubation mixture contained in I.O ml, 25 #moles of Tris-HC1 (pH 7.5), 30 #moles of MgC12, 2.5 #moles of ATP, 0.5/,mole of EDTA, o.oi/,mole of L-~14Clamino acid, 1-2.5 A2e0ms units of t R N A and 0.5 mg of protein. Prior to the addition of enzyme, each solution was incubated at 37 ° for 3 min. The complete mixture was incubated for IO min and then cooled in an ice bath. Two concentrations of t R N A were assayed with each amino acid. Background activity was determined Biochim. Biophys. dcta, 157 (I968) 76-82

E. coli tRNA

79

for each amino acid b y omitting t R N A from the mixture. To measure incorporated radioactivity, the technique described b y BOLLUMTM was followed, and IOO #1 of each reaction were pipetted onto a 2.3 cm W h a t m a n No. 3 MM filter paper disc. Amino acid standard curves were prepared b y pipetting onto discs 5-50/zl of a I : IOOO dilution of L-[14C~amino acid and IOO #1 of a solution containing 5o #g of protein. Dried papers were covered with 5 ml of toluene containing o.oi % dimethyl 1,4-bis-(5phenyloxazolyl-2)-benzene and 0.4 % 2,5-diphenyloxazole and counted for IO rain with a Packard Tri-Carb liquid scintillation counter. Amino acid acceptance activity was calculated as ##moles per A,e 0 ma unit as measured in water. RESULTS

Purification o/the t R N A Tables I and I I summarize data on the purification of tRNA. In Table I, Expt. I reviews data presented in ~ETrlODS. In Expt. II, for the preparation of the crude tRNA, the cells were dispersed in the entire amount of required phenol, and then all of the required water was added. After mixing for I h, this suspension was TABLE

I

PURIFICATION OF t R N A .

RIBCOVERY OF A~60 m# AND YIELDS

F r o z e n cells, (g; 3 ° % s o l i d s ) S t e p i , t R N A , (A860 m~) S t e p 2, t R N A ( % ) S t e p 3, t R N A ( % ) Y i e l d ( g / k g o f E. coli) A260 m ~ / m g

TABLE

Expt. I

Expt. II

Expt. I I I

227 7 2 . 0 " lO 8 43.6 23.0 3.7 °

454 72. 5 • lO 8 34.2 31.o 2.42 20. 4

226 7 0 . 5 • lO 8 32. 4 26. 4 3.95 20.6

19.7

II

PURIFICATION

Fraction

OF tRNA.

RECOVERY

OF AMINO

ACID A C C E P T A N C E

Total weight (g)

A86o mu/mg (units)

Activity [or leucine Speci[ic (t*ttmoles/A,6o my)

Total (tz#moles)

4.914 2.945 1.477 o.286 i.ioo

14. 3 15.7 16.8 11.7 20. 4

46.8 * 1.o8 13o 38 123

3 2 8 0 . 1 0 3* 5 ° . 103 3 2 3 ° . lO 8 127 • lO 8 276o. io 8

7.085 4.461 1.373 -0.900

9.96 lO.5 16.6 -20.6

45.6" 2.8 14o -i63

3 2 1 3 • i o a* 13 • lO 8 3 2 0 0 • lO 8 9 o " I 0 8** 3 0 1 0 • lO 8

Expt. II Step Step Step Step Step

I, 2, 2, 3, 3,

tRNA residue tRNA residue tRNA

Expt. 11I Step Step Step Step Step

I, 2, 2, 3, 3,

tRNA residue tRNA residue tRNA

* Calculated from Step 2 data. ** C a l c u l a t e d f r o m S t e p 2 a n d S t e p 3 d a t a .

Biochim. Biophys. Acta, 157 ( 1 9 6 8 ) 7 6 - 8 2

80

s, GUTCH()

allowed to separate overnight. The purification steps with dimethyl sulfoxide were as described. The low yield associated with this simplified technique was also apparent in later experiments. In Expt. I I I , an equal volume of dimethyl sulfoxide and an equal volume of 3 M NaC1 were taken in the first dimethyl sulfoxide purification. Variation in the volumes of these reagents do not affect either yield or purity. Minimum volumes are those which will effectively clarify the extremely turbid solution of the crude tRNA. Excessive amounts of dilnethyl sulfoxide as well as excessive chilling will precipitate tRNA. The distribution of absorbance into various fractions is not indicative of the quantitative nature of a procedure. The effectiveness of the purification of a crude t R N A can be measured accurately in terms of amino acid acceptance. In Table II, Expts. I I and I I I demonstrate the recovery of activity for leucine. The activity tor crude t R N A (Step I) was calculated from subsequent data since this fraction was isolated prior to the stripping of bound amino acids. The overall recovery of activity was 84.o % and 9 4 . 1 % for Expts. I I and I I I , respectively. Although not tabulated, the distribution of phenylalanine activity was determined, and the corresponding overall recoveries were found to be 81.6 % and 96.1%. Therefore, while a major part of the absorbance of crude t R N A was fractionated into the residues (Table I) only minor parts of these acceptance activities were lost in these residues.

Amino acid acceptance activity Specific activities (/~#moles per A~0 m F in water) of the t R N A recovered from E. coli utilized in the description of the method were as follows: alanine, 55; aspartic 10,0

9.0

8.0

7.O

6.0

=-.5.O E

o

eJ

'~ ,4..0

3.0

2.C

1.0

100

200

300 400 Volume (mr)

500

600

700

Fig. I. Gel f i l t r a t i o n of t R N A on S e p h a d e x G - i o o . t R N A (53 rag) in i M NaC1 w a s c h r o m a t o g r a p h e d on a c o l u m n of S e p h a d e x (2.5 cm × 92 cm) a t a flow r a t e of 3 ° ml / h. E q u i l i b r a t i o n a n d e l u t i o n w e r e w i t h 1 M NaC1 a t 2 °. F r a c t i o n s of 20 m l w e r e collected.

Biochim. Biophys. Acta, 157 (1968) 76-82

E. coli tRNA

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acid, 45; glutamic acid, 48; leucine, 95; phenylalanine, 47; proline, 39; serine, 29; and valine, 7o. As tRNA was isolated from other batches of cells, variations in activities were apparent as may be seen for the leucine values given in Table II and above.

Additional properties o/ the t R N A tRNA prepared as described above showed less than o.5 % DNA 11, negative test for protein b y the biuret reaction lz, absence of nuclease activity 13, sedimentation coefficient (s~0,w) of approx. 4.0, and a lO-15 % decrease in Ao.e0m~ when measured in o.I M Tris chloride (pH 7.5) containing 0.02 M MgC12instead of water. Gel filtration on Sephadex G-ioo (Fig. I) showed a distribution of the A2s0 m~ into 2 peaks with the major, more retarded peak containing 93.5 % of the eluted A260mv; 93.7 % of the added A 2e0m# was recovered from the gel.

DISCUSSION

In order to obtain large amounts of tRNA, the method has been applied to 45 kg batches of E. coli. The recovery of the aqueous phase without any requirement for high speed centrifugation was then of prime importance. The extended time of treatment of the cells in phenol and water was in contrast to the extremely brief exposures described b y BRUBAKER AND McCoRQUODALE 14 and resulted in the recovery of a considerable amount of the non-tRNA components of the E. coli. The subsequent purification of the crude tRNA was achieved readily and fairly quantitatively with respect to the tRNA by the use of dimethyl sulfoxide and NaC1. In addition to a flexibility in the concentration of these reagents, the applicability of other highly polar solvents was determined. In particular, dimethylformamide and dimethylacetamide have been substituted for dimethyl sulfoxide. KIRBY15has reported that dimethyl sulfoxide can be used in place of 2-methoxyethanol in the two-phase separation of high-molecular-weight RNA from glycogen. RNA was extracted into the organic phase. No other application of dimethyl sulfoxide in the purification of a nucleic acid has been noted. A role for dimethyl sulfoxide as a solvent for the purification of a tRNA has been presented above for the first time.

ACKNOWLEDGEMENTS

I would like to thank Louis LAUFER and DAVID R. SCHWARZfor their interest and advice during the above work.

REFERENCES i G. L. BROWN, in J. N. DAVIDSON AND W. E. COHN, Progress in Nucleic Acid Research, Vol. II, A c a d e m i c Press, N e w York, 1963, p. 259. 2 E. J, OFENGAND, M. DIECKMANN AND P. BERG, J. Biol. Chem., 236 (1961) 1741. 3 D. H. RAMMLER, T. OKABAYASHI AND A. DELK, Biochemistry, 4 (I965) 19944 K. S. KIRBY, Biochem. J., 64 (1956) 4o5 .

Biochim. Biophys. Acta, 157 (1968) 76-82

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s. GUTCHO

5 6 7 8 9

R. MONIER, M. L. STEPHENSON AND P. C. ZAMECNIK, Biochim. Biophys. Acta, 43 (19 ~°) J. R. W. HOLLEY, Biochem. Biophys. Res. Commun., IO (1963) 186. M. L. STEPHENSON AND P. C. ZAMECNIK, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1627. R. M. HOSKINSON AND H. G. KHORANA, J. Biol. Chem., 24 ° (1965) 2129. P. BERG, F. H. BERGMANN, E. J. OFENGAND AND M. DIECKMANN, J. Biol. Chem., 236 (1961) 1726. F. J. BOLLUM, in G. L. CANTONI AND D. R. DAVIES, Procedures in Nucleic Acid Research, H a r p e r a n d Row, N e w York, 1966, p. 296. P. I~. STUMPF, J. Biol. Chem., 169 (1947) 367 • M. HOLDEN AND •. W*. PIRIE, Biochem. J., 6o (1955) 46. R. W. HOLLEY, J. APGAR AND S. H. MERRILL, J. Biol. Chem., 236 (I96I) PC42. L. H. BRUBAKER AND D. J. McCORQUODALE,Bioehim. Biophys. Acta, 76 (1963) 48. K. S. KIRBY, Biochim. Biophys. Acta, 55 (1962) 545.

1o II 12 13 14 15

Biochim. Biophys. Acta, 157 (1968) 76-82