- Email: [email protected]
Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus
ANALYTICAL
BIOCHEMISTRY
151,
Two-Step
5
15-5 19 (1985)
Purification of Ttyptophan-Accepting from Bacillus stearothermophilus’
CHARLESW.CARTER,JR.,*,~
DAVIDC.GREEN,* ANDLAURIEBE~S*
tRNA
CLAUDIAS.TOOMIM,-~
Departments of *Biochemistry and TAnatomy, University of North Carolina, Chapel Hill, North Carolina 27514 Received April 29, 1985 Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus. Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [‘4C]tryptophan per A 260unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNAtV were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNAVP has been crystallized under several different conditions. o 1985 Academic press, IIIC. KEY WORDS: tRNA/chromatography of nucleic acids: nucleic acid structure.
Tryptophan-accepting tRNA is among the least abundant of procaryotic tRNA species, and it therefore poses a substantial purification problem. Although this tRNA has been purified to homogeneity from Escherichia coli (1,2), it has not been purified from Bacillus stearothermophilus.We are currently studying the X-ray crystal structure of the tryptophanyltRNA synthetase from the latter organism. Despite the extensive cross-reactivity to heterologous tRNAtV from E. coli (B. R. Reid, personal communication), we wish to study interactions between this enzyme and its homologous cognate tRNA. By combining two of the most frequently used tRNA purification methods we can prepare a high yield of homogeneous tRNAtV from B. stearothermophilus that is suitable for crystallization. There is considerably less diversity among the various methods commonly in use for preparative isolation of pure tRNAs than exists for protein purification procedures. Most methods rely heavily on one or more of the following: chromatography on benzoylated ’ This work was supported by the National Institutes of Health (Grant GM 26203). 515
DEAE-cellulose (BD2-cellulose) (3,4), on DEAE-Sephadex A-50 (5,6), on reversedphase resins (7, lo), and on Sepharose 4B using a reverse salt gradient (11). Preparative aminoacylation ( 1) has a substantial effect on the elution behavior of many tRNAs on most of these chromatographic systems, and has been useful in many purification schemes (1,2,4,9,12). Goss and Parkhurst (12) have used preparative aminoacylation in a singlestep affinity method for all 20 acceptor activities. Joseph and Muench (2) have purified E. coli tRNAtV by chromatographing twice on BD-cellulose with preparative aminoacylation between
runs.
Preparative
aminoacylation
is
a tricky procedure requiring careful choices of ATP, tRNA, and enzyme concentrations, and involves a costly requirement for the cognate tRNA synthetase. The experience of Joseph and Muench (2) suggests that considerable en’ Abbreviations used are: BD-cellulose, benzoylated diethylaminoethyl cellulose; AzM) unit, that quantity of absorbing material which in 1 ml has an optical density of 1 in a l-cm absorbance path; RPC-5 a reversed-phase chromatographic adsorbant prepared as described by Kelmers et al. (7). 0003-2697/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
516
CARTER
ET AL.
richment for tRNA’* from crude tRNA is achieved on the first pass through BD-cellulose and that a suitable second step might be found among other commonly used methods, thereby avoiding preparative aminoacylation. We report here that one species of B. stearothermophilus tRNA@ elutes in a very favorable position from BD-cellulose. Subsequent reverse salt gradient elution from Sepharose. 4B (11) produces high specific activity tRNAtV in good yield. VOLUME(L)
MATERIALS
AND METHODS
Crude tRNA. B. stearothermophilus (NCA 1503) cells were grown for us by E. F. Phares and G. D. Novelli at Oak Ridge National Iaboratory. Crude tRNA was prepared from frozen cells by phenol extraction and salt gradient elution from DEAE-cellulose as described (7). A 4.5 X 60-cm column was filled with a 930ml bed of Whatman DEAE-cellulose (DE-52) equilibrated with Tris-HCl (0.02 M), NaCl (0.25 M), M&l2 (0.01 M), EDTA (0.001 M), Na2S203 (0.002 M), isoamyl acetate (0.1 ml/ liter) pH 7.5. Phenol-extracted and twiceethanol-precipitated nucleic acids were applied at AzeO = 113 (200,800 AzeOunits total). The column was eluted with a 16-liter gradient (0.25-0.65 M NaCl in application buffer) at 24 ml/cm’/h. Tryptophan acceptor activity was assayed by the aminoacylation assay described by Joseph and Muench (2) involving
1 p”‘1
I
^ Ed. x+ c s ei. Em c 2 -<
4. N : ‘1
rd.
I
-
;; D
i-
% zd-t
0.0
-
l:o
2:o
$0 4:o VOLUME
5:o (L)
ST0
7:o
810
FIG. 1. DE-52-cellulose chromatography of crude tRNA from B. stearothermophilus (4°C). The column was developed at 380 ml/h as described in the text. Fractions of 160 ml were collected.
FIG. 2. BD-cellulose chromatography oftRNA(23°C). The column was developed at 130 ml/h and 230-m] fractions were collected.
preincubation of the tRNA with 2.5 mM chloroquine. All buffers and glassware used in the following procedures were autoclaved before use. BD-cellulose chromatography (2). Ethanol pellets of tRNA from two DE-52 columns were combined and dissolved to AZ60 = 200 in sodium acetate (0.01 M), NaCl (0.4 M), MgCl* (0.01 M), EDTA (0.001 M), Na2S203 (0.002 M), pH 5.0. This sample was applied at 23°C (130 AzhO units/ml of column) to a 950-ml, 500 g bed of BD-cellulose (Boehringer-Mannheim) in the same buffer in a 4.5 X 60-cm column. tRNA was eluted with a 7.5-liter NaCl gradient (0.4-l. 1 M) followed by a 3-liter ethanol gradient (0 to 15%) in 1.1 M NaCl at about 8.6 ml/cm*/hr (2). Fractions containing tRNAtP were recovered by ethanol precipitation. A second batch of DE-52-eluted tRNA ( 125,000 A260 units from 1 kg cells) was chromatographed on BD-cellulose (632-g bed, 1200 ml in a 5.2 X 57-cm column). tRNAtn’ was eluted as described previously except that the NaCl gradient was followed by a 2.3-liter wash with 1.1 M NaCl in column buffer before the ethanol gradient was started. Sepharose 4B chromatography (11). Sepharose 4B (Pharmacia) was equilibrated at 4°C with the same buffer used in the previous step (at pH 4.5) with 1.4 M (NH4)*S04 replacing 0.4 M NaCl, and was poured into a 1.6
CHROMATOGRAPHIC
PURIFICATION
OF B. steurothermophilus
517
tRNAtW
to a 1.6 X 78-cm (160-ml) Sepharose 4B column in a slightly different way to avoid uneven packing of the precipitated tRNA. The sample (25 ml, precipitated as above in 1.4 M (NH.&S04) was mixed directly with the top 10 cm of the Sepharose resin and packed uniformly onto the top of the column with 90 ml of 1.4 M (NH&SO4 column buffer. A 1. l-liter reverse salt gradient (1.4-0.0 M (NH&SOJ was pumped through at a rate of 5 ml/cm2/h. VOLUME(L) Fractions containing tRNA’were precipiFIG. 3. Reverse salt gradient elution of tRNAW from tated at 4°C in 67% ethanol. The pellet was Sepharose 4B (4°C). The column was developed at redissolved in distilled, deionized water and IO ml/h and l6-ml fractions were collected. concentrated several times by ultrafiltration (Amicon; PM-lo, 47-mm membrane) to reX 74-cm column to a settled bed volume of move salts and precipitated with ethanol. This 150 ml. tRNA from the previous step was dis- material was denoted recovered tRNA. solved in buffer (plus 1.4 M (NH&304) to AZM) tRNAtrp Crystallization. Conditions for 34 = 400 (30 AZ60 units/ml of column bed) at crystallization trials with purified tRNAtW were 23°C and applied to the column at 4°C. A chosen by the criteria for incomplete factorial heavy precipitate of tRNA formed during the design ( 13). Initial search conditions sampled application, and packed onto the top of the two precipitants (ammonium sulfate and column bed. tRNA from the second BD-celethanol), two temperatures (4 and 14”(Z), three lulose column (4925 AZ60 units) was applied pH values (5.5,6.5, and 7.5), two monovalent TABLE I SUMMARY
OF tRNAw
PURIFICATIONS
Total tRNAVP in Total AzM)
peak
peak
fractions
fraction (nmol)
200,000 270,000
132,766 189,300
1076 2840
0.008 0.015
28 74
8182 lo/84
132,766 124,125
4,680 5,231
430 1400
0.09 0.27
II 36
8182
4,380
394
344”
0.88“
9
IO/84
4,930
612
741
I.21
19
II7 210
254 427
2.18 2.03
I II
Date
Total AZ@ applied
DE-52 (NaCl) 0.25-0.65 M
8182 IO/84
BD Cellulose (NaCI) 0.4-I. I M (Ethanol) 0- 15% Sepharose 4B (Reverse salt gradient) ((NH&S04) 1.4-0.0 M Recovered tRNAlmb
Specific activity nmol
8182 IO/84
A2a
Total tRNA’=’ (mgf
’ Presence of ammonium sulfate in column fractions tends to decrease the tRNA’v acceptor activity. This fully accounts for the increase in specific activity after recovery step. * Only the center of the Sepharose 4B peak was recovered and assayed.
518
CARTER
cations (250 mM Naf or K+), and the presence or absence of 2.5 mM chloroquine, 5 mM Co2’, 50 IIIM acetate, 2 mM Na2S203, 50 mM PO:-, and an equimolar (to tRNA) amount of purified tryptophanyl-tRNA synthetase from B. stearothermophilus (14). All trials contained 5 mg/ml tRNAm, 5 mM MgC12, and 2.5 mM spermine. RESULTS
Gradient elution of tRNA from DE-52 cellulose produced a small but probably significant enrichment for tRNAW (Fig. 1) beyond that which could have been achieved using the customary batch elution. This enrichment may have contributed to the high performance of the two succeeding steps. In contrast to the finding of Joseph and
ET AL.
Muench (2) that E, coli tRNAtW elutes in a region of high general tRNA concentration, tRNAtV from B. stearothermophilus elutes in a region of minimal A260 (Fig. 2). This promising result encouraged us to follow this step with chromatography on Sepharose 4B, using a reverse salt gradient (11). Behavior of tRNAs on this system is determined by differential solubility in (NH4)2S04 and by affinity for the polyol (Sepharose) phase. It should therefore effect a quite different separation of different tRNAs. Reverse salt gradient elution from Sepharose 4B resolved five separate peaks (Fig. 3), the highest of which contained about 80% of the tryptophan acceptor activity applied to the column. The pooled tRNAtW appears to be nearly homogeneous, having an acceptor activity
FIG. 4. Crystals grown from solutions (5 mg/ml) of purified B. steurothermophilus tRNA’m. (a-c) Crystals of tRNAtm grown by vapor diffusion against 90% saturated ammonium sulfate. (d) Crystals grown in the presence of 7.1 mg/ml of B. stearothermophilur tryptophanyl-tRNA synthetase in addition to tRNA-. Final concentration of ammonium sulfate was 70%.
CHROMATOGRAPHIC
PURIFICATION
close to that expected for the pure species (1800 pmol/f&m unit; (2)). The last two entries in Table 1 represent determinations of this value for recovered tRNA from the two prep arations. These had acceptor activities of 2200 and 2000 prr~ol/A~~~ unit. A subsequent chromatography step on RPC 5 (&lo) did not alter this value appreciably. Two different purifications using this scheme are summarized in Table 1. BD-cellulose chromatography yields between 50 and 75% of the total tryptophan acceptor activity in the major peak; the better yield and higher specific activity in the second trial results from the extra washing step (one column vol 1.1 M NaCl) prior to the ethanol gradient (Fig. 2). Most of the rest of the acceptor activity is accounted for in two small peaks at 7.0 and 15.5 liters elution volume, respectively. We have not determined whether or not these reproducible peaks may be isoaccepting species of tRNA”“. This procedure yields approximately a milligram of pure tRNA from 100 g frozen cells. This yield compares favorably with that achieved for other procaryotic tRNAs (B. R. Reid, personal communication). The purity of the tRNAtm is further demonstrated by the fact that it crystallizes under several different conditions. Figures 4 a-d shows results of crystallization trials with purified tRNAtV. These have not been characterized beyond showing that they crush easily and hence that they are macromolecular. The crystals in Fig. 4d grew in the presence of an equimolar amount of Bacillus steurothermophitus tryptophanyl-tRNA synthetase (14). It is not known whether the crystals contain a complex of the synthetase and its tRNA, or are made up solely of one or the other component. A complete analysis of the incomplete factorial
OF B. steurothermophilus
519
tRNAm
results from all experimental presently underway.
conditions
is
ACKNOWLEDGMENTS We thank Brian R. Reid for his advice concerning several phases of this work, and E. F. Phares and G. D. NoveIli (deceased) for their generous gift of B. steurofhermophilus cells.
REFERENCES Muench, K. H. (1971) Methods Mol. Biol. 1, 235265. Joseph, D. R., and Muench, K. H. (1971) J. Biol. Chem. 246,7610-7615. Gillam, I. C. and Tener, G. M. (197 1) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, Part C, pp. 20, 55-70, Academic Press, New York. 4. Gillam, I. C., Blew, D., Warrington, R. C., von Tigerstrom, M., and Tener, G. M. (1968) Biochemisfry I, 3459-3461. 5. Reid, B. R., Ribeiro, N. S., McCollum, L., Abbate, J., and Hurd, R. E. (1977) Biochemistry 16,20862094. 6. Nishimura, S. (197 1) Proc. Nucl. Acids Res. 2, 542564. 7. Kelmers, A. D., Hancher, C. W., Phares, E. F., and Novelli, G. D. (1974) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, Part C, pp. 20,3-9, Academic Press, New York. 8. Waters, L. C., and Novelli, G. D. (197 I) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, Part C, pp. 20, 39-44, Academic Press, New York. Waters, L. C., Yang, W.-K, Mulhn, B. C., and Nichols, J. L. (1975) J. Biol. Chem. 250,6627-6629. Kelmers, A. D., and Heatherly, D. E. (1971). Anal. Biochem. 44,486-495. Holmes, W. M., Hurd, R. E., Reid, B. R., Limmerman, R. A., and Hatfield, G. W. (1975) Proc. Natl. Acad. Sci. USA 72, 1068-1071. Goss, D. J., and Parkhurst, L. J. ( 1978) J. Biol. Chem. 253,7804-7806. Carter, C. W., Jr., and Carter, C. W. (1979) J. Biol. Chem. 254, 12219-12223. Carter, C. W., Jr., and Green, D. C. (1982) Anal. Biochem. 124,321-332.
BIOCHEMISTRY
151,
Two-Step
5
15-5 19 (1985)
Purification of Ttyptophan-Accepting from Bacillus stearothermophilus’
CHARLESW.CARTER,JR.,*,~
DAVIDC.GREEN,* ANDLAURIEBE~S*
tRNA
CLAUDIAS.TOOMIM,-~
Departments of *Biochemistry and TAnatomy, University of North Carolina, Chapel Hill, North Carolina 27514 Received April 29, 1985 Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus. Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [‘4C]tryptophan per A 260unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNAtV were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNAVP has been crystallized under several different conditions. o 1985 Academic press, IIIC. KEY WORDS: tRNA/chromatography of nucleic acids: nucleic acid structure.
Tryptophan-accepting tRNA is among the least abundant of procaryotic tRNA species, and it therefore poses a substantial purification problem. Although this tRNA has been purified to homogeneity from Escherichia coli (1,2), it has not been purified from Bacillus stearothermophilus.We are currently studying the X-ray crystal structure of the tryptophanyltRNA synthetase from the latter organism. Despite the extensive cross-reactivity to heterologous tRNAtV from E. coli (B. R. Reid, personal communication), we wish to study interactions between this enzyme and its homologous cognate tRNA. By combining two of the most frequently used tRNA purification methods we can prepare a high yield of homogeneous tRNAtV from B. stearothermophilus that is suitable for crystallization. There is considerably less diversity among the various methods commonly in use for preparative isolation of pure tRNAs than exists for protein purification procedures. Most methods rely heavily on one or more of the following: chromatography on benzoylated ’ This work was supported by the National Institutes of Health (Grant GM 26203). 515
DEAE-cellulose (BD2-cellulose) (3,4), on DEAE-Sephadex A-50 (5,6), on reversedphase resins (7, lo), and on Sepharose 4B using a reverse salt gradient (11). Preparative aminoacylation ( 1) has a substantial effect on the elution behavior of many tRNAs on most of these chromatographic systems, and has been useful in many purification schemes (1,2,4,9,12). Goss and Parkhurst (12) have used preparative aminoacylation in a singlestep affinity method for all 20 acceptor activities. Joseph and Muench (2) have purified E. coli tRNAtV by chromatographing twice on BD-cellulose with preparative aminoacylation between
runs.
Preparative
aminoacylation
is
a tricky procedure requiring careful choices of ATP, tRNA, and enzyme concentrations, and involves a costly requirement for the cognate tRNA synthetase. The experience of Joseph and Muench (2) suggests that considerable en’ Abbreviations used are: BD-cellulose, benzoylated diethylaminoethyl cellulose; AzM) unit, that quantity of absorbing material which in 1 ml has an optical density of 1 in a l-cm absorbance path; RPC-5 a reversed-phase chromatographic adsorbant prepared as described by Kelmers et al. (7). 0003-2697/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
516
CARTER
ET AL.
richment for tRNA’* from crude tRNA is achieved on the first pass through BD-cellulose and that a suitable second step might be found among other commonly used methods, thereby avoiding preparative aminoacylation. We report here that one species of B. stearothermophilus tRNA@ elutes in a very favorable position from BD-cellulose. Subsequent reverse salt gradient elution from Sepharose. 4B (11) produces high specific activity tRNAtV in good yield. VOLUME(L)
MATERIALS
AND METHODS
Crude tRNA. B. stearothermophilus (NCA 1503) cells were grown for us by E. F. Phares and G. D. Novelli at Oak Ridge National Iaboratory. Crude tRNA was prepared from frozen cells by phenol extraction and salt gradient elution from DEAE-cellulose as described (7). A 4.5 X 60-cm column was filled with a 930ml bed of Whatman DEAE-cellulose (DE-52) equilibrated with Tris-HCl (0.02 M), NaCl (0.25 M), M&l2 (0.01 M), EDTA (0.001 M), Na2S203 (0.002 M), isoamyl acetate (0.1 ml/ liter) pH 7.5. Phenol-extracted and twiceethanol-precipitated nucleic acids were applied at AzeO = 113 (200,800 AzeOunits total). The column was eluted with a 16-liter gradient (0.25-0.65 M NaCl in application buffer) at 24 ml/cm’/h. Tryptophan acceptor activity was assayed by the aminoacylation assay described by Joseph and Muench (2) involving
1 p”‘1
I
^ Ed. x+ c s ei. Em c 2 -<
4. N : ‘1
rd.
I
-
;; D
i-
% zd-t
0.0
-
l:o
2:o
$0 4:o VOLUME
5:o (L)
ST0
7:o
810
FIG. 1. DE-52-cellulose chromatography of crude tRNA from B. stearothermophilus (4°C). The column was developed at 380 ml/h as described in the text. Fractions of 160 ml were collected.
FIG. 2. BD-cellulose chromatography oftRNA(23°C). The column was developed at 130 ml/h and 230-m] fractions were collected.
preincubation of the tRNA with 2.5 mM chloroquine. All buffers and glassware used in the following procedures were autoclaved before use. BD-cellulose chromatography (2). Ethanol pellets of tRNA from two DE-52 columns were combined and dissolved to AZ60 = 200 in sodium acetate (0.01 M), NaCl (0.4 M), MgCl* (0.01 M), EDTA (0.001 M), Na2S203 (0.002 M), pH 5.0. This sample was applied at 23°C (130 AzhO units/ml of column) to a 950-ml, 500 g bed of BD-cellulose (Boehringer-Mannheim) in the same buffer in a 4.5 X 60-cm column. tRNA was eluted with a 7.5-liter NaCl gradient (0.4-l. 1 M) followed by a 3-liter ethanol gradient (0 to 15%) in 1.1 M NaCl at about 8.6 ml/cm*/hr (2). Fractions containing tRNAtP were recovered by ethanol precipitation. A second batch of DE-52-eluted tRNA ( 125,000 A260 units from 1 kg cells) was chromatographed on BD-cellulose (632-g bed, 1200 ml in a 5.2 X 57-cm column). tRNAtn’ was eluted as described previously except that the NaCl gradient was followed by a 2.3-liter wash with 1.1 M NaCl in column buffer before the ethanol gradient was started. Sepharose 4B chromatography (11). Sepharose 4B (Pharmacia) was equilibrated at 4°C with the same buffer used in the previous step (at pH 4.5) with 1.4 M (NH4)*S04 replacing 0.4 M NaCl, and was poured into a 1.6
CHROMATOGRAPHIC
PURIFICATION
OF B. steurothermophilus
517
tRNAtW
to a 1.6 X 78-cm (160-ml) Sepharose 4B column in a slightly different way to avoid uneven packing of the precipitated tRNA. The sample (25 ml, precipitated as above in 1.4 M (NH.&S04) was mixed directly with the top 10 cm of the Sepharose resin and packed uniformly onto the top of the column with 90 ml of 1.4 M (NH&SO4 column buffer. A 1. l-liter reverse salt gradient (1.4-0.0 M (NH&SOJ was pumped through at a rate of 5 ml/cm2/h. VOLUME(L) Fractions containing tRNA’were precipiFIG. 3. Reverse salt gradient elution of tRNAW from tated at 4°C in 67% ethanol. The pellet was Sepharose 4B (4°C). The column was developed at redissolved in distilled, deionized water and IO ml/h and l6-ml fractions were collected. concentrated several times by ultrafiltration (Amicon; PM-lo, 47-mm membrane) to reX 74-cm column to a settled bed volume of move salts and precipitated with ethanol. This 150 ml. tRNA from the previous step was dis- material was denoted recovered tRNA. solved in buffer (plus 1.4 M (NH&304) to AZM) tRNAtrp Crystallization. Conditions for 34 = 400 (30 AZ60 units/ml of column bed) at crystallization trials with purified tRNAtW were 23°C and applied to the column at 4°C. A chosen by the criteria for incomplete factorial heavy precipitate of tRNA formed during the design ( 13). Initial search conditions sampled application, and packed onto the top of the two precipitants (ammonium sulfate and column bed. tRNA from the second BD-celethanol), two temperatures (4 and 14”(Z), three lulose column (4925 AZ60 units) was applied pH values (5.5,6.5, and 7.5), two monovalent TABLE I SUMMARY
OF tRNAw
PURIFICATIONS
Total tRNAVP in Total AzM)
peak
peak
fractions
fraction (nmol)
200,000 270,000
132,766 189,300
1076 2840
0.008 0.015
28 74
8182 lo/84
132,766 124,125
4,680 5,231
430 1400
0.09 0.27
II 36
8182
4,380
394
344”
0.88“
9
IO/84
4,930
612
741
I.21
19
II7 210
254 427
2.18 2.03
I II
Date
Total AZ@ applied
DE-52 (NaCl) 0.25-0.65 M
8182 IO/84
BD Cellulose (NaCI) 0.4-I. I M (Ethanol) 0- 15% Sepharose 4B (Reverse salt gradient) ((NH&S04) 1.4-0.0 M Recovered tRNAlmb
Specific activity nmol
8182 IO/84
A2a
Total tRNA’=’ (mgf
’ Presence of ammonium sulfate in column fractions tends to decrease the tRNA’v acceptor activity. This fully accounts for the increase in specific activity after recovery step. * Only the center of the Sepharose 4B peak was recovered and assayed.
518
CARTER
cations (250 mM Naf or K+), and the presence or absence of 2.5 mM chloroquine, 5 mM Co2’, 50 IIIM acetate, 2 mM Na2S203, 50 mM PO:-, and an equimolar (to tRNA) amount of purified tryptophanyl-tRNA synthetase from B. stearothermophilus (14). All trials contained 5 mg/ml tRNAm, 5 mM MgC12, and 2.5 mM spermine. RESULTS
Gradient elution of tRNA from DE-52 cellulose produced a small but probably significant enrichment for tRNAW (Fig. 1) beyond that which could have been achieved using the customary batch elution. This enrichment may have contributed to the high performance of the two succeeding steps. In contrast to the finding of Joseph and
ET AL.
Muench (2) that E, coli tRNAtW elutes in a region of high general tRNA concentration, tRNAtV from B. stearothermophilus elutes in a region of minimal A260 (Fig. 2). This promising result encouraged us to follow this step with chromatography on Sepharose 4B, using a reverse salt gradient (11). Behavior of tRNAs on this system is determined by differential solubility in (NH4)2S04 and by affinity for the polyol (Sepharose) phase. It should therefore effect a quite different separation of different tRNAs. Reverse salt gradient elution from Sepharose 4B resolved five separate peaks (Fig. 3), the highest of which contained about 80% of the tryptophan acceptor activity applied to the column. The pooled tRNAtW appears to be nearly homogeneous, having an acceptor activity
FIG. 4. Crystals grown from solutions (5 mg/ml) of purified B. steurothermophilus tRNA’m. (a-c) Crystals of tRNAtm grown by vapor diffusion against 90% saturated ammonium sulfate. (d) Crystals grown in the presence of 7.1 mg/ml of B. stearothermophilur tryptophanyl-tRNA synthetase in addition to tRNA-. Final concentration of ammonium sulfate was 70%.
CHROMATOGRAPHIC
PURIFICATION
close to that expected for the pure species (1800 pmol/f&m unit; (2)). The last two entries in Table 1 represent determinations of this value for recovered tRNA from the two prep arations. These had acceptor activities of 2200 and 2000 prr~ol/A~~~ unit. A subsequent chromatography step on RPC 5 (&lo) did not alter this value appreciably. Two different purifications using this scheme are summarized in Table 1. BD-cellulose chromatography yields between 50 and 75% of the total tryptophan acceptor activity in the major peak; the better yield and higher specific activity in the second trial results from the extra washing step (one column vol 1.1 M NaCl) prior to the ethanol gradient (Fig. 2). Most of the rest of the acceptor activity is accounted for in two small peaks at 7.0 and 15.5 liters elution volume, respectively. We have not determined whether or not these reproducible peaks may be isoaccepting species of tRNA”“. This procedure yields approximately a milligram of pure tRNA from 100 g frozen cells. This yield compares favorably with that achieved for other procaryotic tRNAs (B. R. Reid, personal communication). The purity of the tRNAtm is further demonstrated by the fact that it crystallizes under several different conditions. Figures 4 a-d shows results of crystallization trials with purified tRNAtV. These have not been characterized beyond showing that they crush easily and hence that they are macromolecular. The crystals in Fig. 4d grew in the presence of an equimolar amount of Bacillus steurothermophitus tryptophanyl-tRNA synthetase (14). It is not known whether the crystals contain a complex of the synthetase and its tRNA, or are made up solely of one or the other component. A complete analysis of the incomplete factorial
OF B. steurothermophilus
519
tRNAm
results from all experimental presently underway.
conditions
is
ACKNOWLEDGMENTS We thank Brian R. Reid for his advice concerning several phases of this work, and E. F. Phares and G. D. NoveIli (deceased) for their generous gift of B. steurofhermophilus cells.
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