[39]
tRNA SPLICINGENDONUCLEASEFROMS. cerevisiae
471
TABLE I PURIFICATION OF YEAST t R N A LIGASE FROM Escherichia coli OVERPRODUCING STRAIN
Step Crude extract Polymin P supernatant Heparin-agarose Blue Trisacryl M Hydroxyapatite Sephadex G-150
Total volume (ml)
Total protein
Total activity (kU)
700 620
18 g 12.4 g
190 170
360 170 70 100
305 64 42 25
mg mg mg mg
110 70 60 58
Specific activity (U/mg) 10.5 13.7 360 1093 1428 2320
Purification (-fold) 1 1.3 34.3 104 136 220
Recovery (%) 100 89.4 58 37 32 30
Conclusions This procedure provides 25 mg of pure tRNA ligase with a 30% recovery of the activity (Table I). The peak fractions from the final gel filtration step can be concentrated by ultrafiltration and are stored in buffer A containing 0.2 M NaCI and 50% glycerol at -20 °. The main problem that had to be solved in this purification was the sensitivity of the protein to protease. Three measures were taken to prevent degradation: the gene was expressed in a protease-deficient strain, the first three steps of the protocol were done as rapidly as possible, and a mixture of protease inhibitors are included in all of the chromatography steps. The tRNA ligase, as purified, possesses all three of the constituent activities.
[39] H i g h l y P u r i f i e d T r a n s f e r R N A S p l i c i n g E n d o n u c l e a s e from Saccharomyces cerevisiae By PHILLIP R. GREEN and JOHN N. ABELSON Intervening sequences are found in and spliced from various messenger, ribosomal, and transfer RNAs. The first splicing reaction demonstrated in vitro was for tRNA precursors in crude extracts from the yeast Saccharomyces cerevisiae. 1 Isolated labeled tRNA precursors 1'2 are t G. Knapp, J. J. Beckmann, P. F. Johnson, S. A. Fuhrman, and J. Abelson, Cell 14, 221 (1978). 2 A. K. Hopper, F. Banks, and V. Evangelidis, Cell 14, 211 (1978).
METHODS IN ENZYMOLOGY, VOL. 181
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
472
PROCESSINGOF TRANSFERRNAs
[39]
spliced via a two-step mechanism) First, an endonuclease cleaves the precursor at the 5' and 3' splice sites, releasing the linear intron and tRNA half-molecules. The endonuclease cleavage products contain 5'-hydroxyls and 2',3'-cyclic phosphates. 4 In the second step, the tRNA half-molecules are ligated in a complex, ATP-dependent reaction catalyzed by the splicing ligase. 5 Our goal is to study biochemically and genetically tRNA splicing in yeast to determine its possible role in gene expression. One approach is to purify the protein and use sequence data to isolate the gene. This approach has been successfully used for the tRNA splicing ligase. 6 The ligase, a single polypeptide of 90,000 Da, catalyzes all of the reactions involved in the ligation of the cut precursors. The endonuclease has, to this time, been refractory to complete purification. Like the ligase, it is present in small amounts. It also acts as an integral membrane-bound protein. In this chapter, a method used to extensively purify small amounts of the endonuclease is presented. The data suggest that the enzyme may be multimeric. Assay of Activity 3,4
A 32p-labeled synthetic precursor tRNA Phe is used as the substrate. 7 A modified, intron-containing tRNA Pne gene is synthesized de novo. The bacteriophage T7 RNA polymerase promoter is inserted immediately 3' of the gene, and a BstNI restriction endonuclease site is included at the 5' end. The gene is cloned into pUC13, transformed into Escherichia coli strain JM101, and plasmid DNA isolated. The plasmids are cut with BstNI and used to make T7 RNA polymerase runoff transcripts in the presence of [a-32p]UTP. Precursor of about 3 × 106 dpm/pmol is obtained. The reaction is performed in 10/~1 of buffer containing 40 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HC1) (pH 8.0), 0.5 mM ethylenediaminetetraacetic acid, disodium salt (Na2 EDTA), 4.0 mM spermidine-HCl, 0.2% Triton X-100 (w/v), 10% glycerol, and 1 × 10-15 mol (1,000-2,000 cpm) of tRNA precursor. To this buffer, 1 tzl of appropriately diluted [into 10% glycerol (w/v), 0.5% Triton X-100, 25 mM TrisHCI (pH 8.0), and 5 mM 2-mercaptoethanol] endonuclease is added at 0 °, mixed, and incubated for 10 min at 30°. The reaction is stopped by adding 1/zl of a solution containing 2% sodium dodecyl sulfate (SDS), 100 mM 3 C. 4 C. 5 C. 6 E. 7 V.
L. Peebles, R. C. Ogden, G. Knapp, and J. Abelson, Cell 18, 27 (1979). L. Peebles, P. Gegenheimer, and J. Abelson, Cell 32, 525 (1983). L. Greer, C. L. Peebles, P. Gegenheimer, and J. Abelson, Cell 32, 537 (1983). M. Phizicky, R. C. Schwartz, and J. Abelson, J. Biol. Chem. 261, 2978 (1986). Reyes and J. Abelson, Anal. Biochem. 166, 90 (1987).
[39]
tRNA SPLICINGENDONUCLEASEFROMS. cerevisiae
473
EDTA, and 2.0 mg/ml proteinase K and incubated at 50° for 15 min. The products are mixed with 5/zl of 8 M urea, 20% sucrose, bromphenol blue, and xylene cyanol, heated to 65° for 5 rain, and separated by electrophoresis through a 10% polyacrylamide (acrylamide-bisacrylamide, 30: 1) gel containing 90 mM Tris-borate (pH 8.3), 2.5 mM EDTA, and 4 M urea) The 32p-labeled half-molecules are located by autoradiography and cut out of the gels, and the Cerenkov radiation is measured assuming a counting efficiency of 30%. A unit of activity is defined as the amount of enzyme needed to catalyze the cleavage of 1 x 10-12 mol of tRNA precursor.
Protein Determination Samples are precipitated with 5% trichloroacetic acid, 8,9 and the protein concentration is estimated by the method of Lowry et al.,10 using bovine serum albumin as the standard. Solutions contain 1% SDS to prevent interference by Triton X-100.
Electrophoresis Protein samples are precipitated in 1 ml of 5% trichloroacetic acid. After a 5-min centrifugation in a microcentrifuge, Tris base is added to the solution to a concentration of 0.3 M. Triton X-100 redissolves upon mixing, and precipitated protein is isolated by centrifugation for 10 min. The pellet is resuspended in 15/zl of sample buffer plus 2/zl of 2 M Tris base, heated for 3 min at 100°, and separated on a 5-15% polyacrylamide gradient gel according to Laemmli. H Separated proteins are visualized by silver staining) 2
Growth o f Yeast The protease-deficient S. cerevisiae strain 20B-12-1 (a-pep4-3, prcl, prbl, his) 5 is grown to a n A600 of 4-6 at 30° in 350 liters of 1% yeast extract, 2% peptone, and 2% glucose. The yeast are stored at - 7 0 ° until needed.
Purification Scheme Pellets and solutions are stored at - 2 0 °, and all steps are performed at 4° . s A. Bensadoun and D. Weinstein, Anal. Biochem. 70, 241 (1976). 9 p. R. Green, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 256, 11151 (1981). 10 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). it U. K. Laemmli, Nature (London) 227, 680 (1970). i2 W. Wray, T. Boulikas, V. P. Wray, and R. Hancock, Anal. Biochem. 118, 197 (1981).
474
PROCESSINGOF TRANSFERRNAs
[39]
TABLE I PURIFICATION OF YEAST tRNA SPLICING ENDONUCLEASEu
Purification step
Total activity (units)
Total protein (mg)
Specific activity (units/mg)
Purification (-fold)
Yield (%)
Triton X-100 extract Heparin-agarose 1 Heparin-agarose 2 Hydroxyapatite flow-through Hydroxyapatite Affi-Gel Blue tRNA elution
570 767 637 590 513 95 87
5,400 439 135 50 11.6 0.89 0.40 b
0.11 1.8 4.7 11.8 44.2 107 218
1 16 43 107 402 970 1,980
100 135 112 104 90 17 15
a The endonuclease was purified from 3 kg of yeast. Details of the steps employed are described in the text. b Protein was estimated by comparing volumes of the fraction loaded onto the SDSpolyacrylamide gel and the intensity of the stained protein bands between the Affi-Gel Blue and tRNA elution steps (see Fig. 2, lanes 7 and 8).
Preparation of Detergent Extract. Frozen cells are thawed in 2 volumes of 100 m M Tris-HCl (pH 8.0), 20 m M EDTA, 10% glycerol, 5 mM spermidine-HCl, 3 mM dithiothreitol, 1.0 M ammonium sulfate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells are lysed with glass beads (0.20-0.30 mm diameter; Glen Mills, Inc., Maywood, NJ) by passing them twice through a Dynomill chilled at - 1 0 °. The extract is centrifuged at 4,000 g for 10 min and the supernatant at 150,000 g for 2 hr. The membranes are homogenized into 2 volumes of lysis buffer using a Potter-Elvehjem homogenizer and centrifuged as above. The membranes are washed twice further by homogenizing into 20% glycerol, 5 mM 2mercaptoethanol, 25 m M Tris-HC1 (pH 8.0), 0.2 mM PMSF (buffer E), adding 0.02% Triton X-100, and stirring for 1 hr. The washed membranes are homogenized into 2 volumes of buffer E (20-40 mg/ml protein), Triton X-100 is added to 0.7%, and the mixture is stirred for 1 hr and centrifuged. This extract contains 5-50% of the extractable activity. The remaining activity is extracted as above using 0.9% Triton X-100 plus 0.1 M ammonium sulfate. Since activity is difficult to quantitate while membrane bound, the detergent extracts are defined to be 1-fold purified (Table I, Fig. 2, lane 2). The extracts may be stored indefinitely at - 2 0 °. Because of the low yield of endonuclease activity, we find it necessary to process kilogram quantities of yeast. To obtain higher yields of membranes, it is critical to centrifuge a full 2 hr. Still, much activity is found in the milky supernatant, perhaps in very small vesicles.
[39]
tRNA SPLICINGENDONUCLEASEFROMS. cerevisiae
475
Heparin-Agarose Chromatography. The extracts are made 2.5% in Triton X-100 by adding a 20% stock solution. The extract is applied to a 4 × 12 cm column of heparin-agarose that has been equilibrated with buffer C [25 m M Tris-HCl (pH 8.0), 10% glycerol, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100] containing 0.1 M ammonium sulfate. Bound protein is eluted at 150 ml/hr with a 2-liter gradient of 0.1-0.6 M ammonium sulfate in buffer C (Fig. 1A and Fig. 2, lane 3). Active fractions are combined, diluted 3-fold with buffer C, and applied to a 2 × 38 cm heparin-agarose column. Activity is eluted at 70 ml/hr with a l-liter gradient of 0.1-0.6 M ammonium sulfate (Fig. 1B and Fig. 2, lane 4). Hydroxyapatite Chromatography. The active heparin-agarose fractions are combined but cannot be dialyzed without extensive precipitation and loss of activity. They are applied to a 2 x 35 cm column of hydroxyapatite (BioGel HTP, Bio-Rad) equilibrated with buffer C containing 0.35 M ammonium sulfate. Most of the activity flows through the column (Fig. 2, lane 5). The flow-through is collected, diluted 3-fold with buffer C, and reapplied to the hydroxyapatite column equilibrated with buffer C. Activity is eluted at 70 ml/hr with a 1,100-ml gradient of 10% glycerol, 5 m M 2-mercaptoethanol, 0.5% Triton X-100, and 0-0.45 M potassium phosphate (pH 7.0) (Fig. 1C and Fig. 2, lane 6). Affi-Gel Blue Chromatography. The active hydroxyapatite fractions are combined and dialyzed against buffer C until the potassium phosphate concentration is less than 75 mM, then applied to a 1 x 16 cm column of Affi-Gel Blue (Bio-Rad). Activity is eluted at 30 ml/hr using a 1,100-ml gradient of buffer C containing 0-2.0 M NaC1 (Fig. 1D and Fig. 2, lane 7). The yield of activity is consistently low (10-20%). It can be increased by using a smaller column ( - 7 ml); however, this greatly decreases the efficiency in removing certain contaminants. tRNA Affinity Elution. Active fractions from the Affi-Gel Blue step are combined, dialyzed against buffer C, and applied to a 1 x 8 cm column of CM-Sepharose. The column is washed with 25 ml of buffer C containing 75 mM NaC1, and activity is eluted at 40 ml/hr with 100 ml of the same buffer plus 100 mg/ml yeast tRNA (Type X-S, Sigma, treated with proteinase K, phenol extracted, and ethanol precipitated) (Fig. 2, lane 8). Glycerol Gradients. The affinity eluant is concentrated by applying it to a 1-ml column of hydroxyapatite and step eluting with 10 ml of buffer C containing 0.4 M potassium phosphate (pH 7.0). A portion (300 /zl) is layered on an ll-ml, 10-16% glycerol gradient in buffer C plus 0.4 M potassium phosphate (pH 7.0). The gradients are centrifuged at 38,000 rpm (180,000 g) for 48 hr in an SW41 rotor. The purification is summarized in Table I and Fig. 2. The overall purification through the tRNA affinity column is about 2,000-fold over
476
PROCESSING OF TRANSFER R N A s A
/
v
0.6
x
×/× /
2.00
"7
[39]
0.80~
0.5 v
E LSO
0.60 ~
>I - 1.00 > I.,-
Z 0.40 ~
v
0.4. ~
c~ or) O.3 ~
h--
0.20
0.50
13_
0 I
Z 0.2 ' v
,
0.1
I
I
I
20
40
FRACTION
I
I
60
NUMBER
Bf
/x
4.oo
72.oot
tt 0.6 lo. o lo, o.eo "~"
o~
/, ~, ix
0
I
I0
20
FRACTION
30
OI
40
NUMBER
FIG. 1. Elution profiles. (A) Chromatography of Triton X-100 extract over heparinagarose. (B) Second chromatography over heparin-agarose. (C) Chromatography over hydroxyapatite. (D) Chromatography over Affi-Gel Blue. Active fractions combined for the next step are indicated by a bar in the figures.
[39]
tRNA SPLICING ENDONUCLEASE FROM S. cereoisiae
477
O.OeO? ]0.50
?4.o
1o.o io. o
/x x
~[~o
E
'w
1oo,,o~1o.~o~ z
x/
l
,
0
I0
T
13_
,
,
20 :50 40 FRACTION NUMBER
0_
]0
50
2.0
D X
4.0
0.04().~
1.6
0.03([
1.2
0.02( Z
0.8 :~
Q v
X
"~3.0
v
5
v
~ 2.0
P_
> I.~O
tRNA SPLICINGENDONUCLEASEFROMS. cerevisiae
471
TABLE I PURIFICATION OF YEAST t R N A LIGASE FROM Escherichia coli OVERPRODUCING STRAIN
Step Crude extract Polymin P supernatant Heparin-agarose Blue Trisacryl M Hydroxyapatite Sephadex G-150
Total volume (ml)
Total protein
Total activity (kU)
700 620
18 g 12.4 g
190 170
360 170 70 100
305 64 42 25
mg mg mg mg
110 70 60 58
Specific activity (U/mg) 10.5 13.7 360 1093 1428 2320
Purification (-fold) 1 1.3 34.3 104 136 220
Recovery (%) 100 89.4 58 37 32 30
Conclusions This procedure provides 25 mg of pure tRNA ligase with a 30% recovery of the activity (Table I). The peak fractions from the final gel filtration step can be concentrated by ultrafiltration and are stored in buffer A containing 0.2 M NaCI and 50% glycerol at -20 °. The main problem that had to be solved in this purification was the sensitivity of the protein to protease. Three measures were taken to prevent degradation: the gene was expressed in a protease-deficient strain, the first three steps of the protocol were done as rapidly as possible, and a mixture of protease inhibitors are included in all of the chromatography steps. The tRNA ligase, as purified, possesses all three of the constituent activities.
[39] H i g h l y P u r i f i e d T r a n s f e r R N A S p l i c i n g E n d o n u c l e a s e from Saccharomyces cerevisiae By PHILLIP R. GREEN and JOHN N. ABELSON Intervening sequences are found in and spliced from various messenger, ribosomal, and transfer RNAs. The first splicing reaction demonstrated in vitro was for tRNA precursors in crude extracts from the yeast Saccharomyces cerevisiae. 1 Isolated labeled tRNA precursors 1'2 are t G. Knapp, J. J. Beckmann, P. F. Johnson, S. A. Fuhrman, and J. Abelson, Cell 14, 221 (1978). 2 A. K. Hopper, F. Banks, and V. Evangelidis, Cell 14, 211 (1978).
METHODS IN ENZYMOLOGY, VOL. 181
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
472
PROCESSINGOF TRANSFERRNAs
[39]
spliced via a two-step mechanism) First, an endonuclease cleaves the precursor at the 5' and 3' splice sites, releasing the linear intron and tRNA half-molecules. The endonuclease cleavage products contain 5'-hydroxyls and 2',3'-cyclic phosphates. 4 In the second step, the tRNA half-molecules are ligated in a complex, ATP-dependent reaction catalyzed by the splicing ligase. 5 Our goal is to study biochemically and genetically tRNA splicing in yeast to determine its possible role in gene expression. One approach is to purify the protein and use sequence data to isolate the gene. This approach has been successfully used for the tRNA splicing ligase. 6 The ligase, a single polypeptide of 90,000 Da, catalyzes all of the reactions involved in the ligation of the cut precursors. The endonuclease has, to this time, been refractory to complete purification. Like the ligase, it is present in small amounts. It also acts as an integral membrane-bound protein. In this chapter, a method used to extensively purify small amounts of the endonuclease is presented. The data suggest that the enzyme may be multimeric. Assay of Activity 3,4
A 32p-labeled synthetic precursor tRNA Phe is used as the substrate. 7 A modified, intron-containing tRNA Pne gene is synthesized de novo. The bacteriophage T7 RNA polymerase promoter is inserted immediately 3' of the gene, and a BstNI restriction endonuclease site is included at the 5' end. The gene is cloned into pUC13, transformed into Escherichia coli strain JM101, and plasmid DNA isolated. The plasmids are cut with BstNI and used to make T7 RNA polymerase runoff transcripts in the presence of [a-32p]UTP. Precursor of about 3 × 106 dpm/pmol is obtained. The reaction is performed in 10/~1 of buffer containing 40 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HC1) (pH 8.0), 0.5 mM ethylenediaminetetraacetic acid, disodium salt (Na2 EDTA), 4.0 mM spermidine-HCl, 0.2% Triton X-100 (w/v), 10% glycerol, and 1 × 10-15 mol (1,000-2,000 cpm) of tRNA precursor. To this buffer, 1 tzl of appropriately diluted [into 10% glycerol (w/v), 0.5% Triton X-100, 25 mM TrisHCI (pH 8.0), and 5 mM 2-mercaptoethanol] endonuclease is added at 0 °, mixed, and incubated for 10 min at 30°. The reaction is stopped by adding 1/zl of a solution containing 2% sodium dodecyl sulfate (SDS), 100 mM 3 C. 4 C. 5 C. 6 E. 7 V.
L. Peebles, R. C. Ogden, G. Knapp, and J. Abelson, Cell 18, 27 (1979). L. Peebles, P. Gegenheimer, and J. Abelson, Cell 32, 525 (1983). L. Greer, C. L. Peebles, P. Gegenheimer, and J. Abelson, Cell 32, 537 (1983). M. Phizicky, R. C. Schwartz, and J. Abelson, J. Biol. Chem. 261, 2978 (1986). Reyes and J. Abelson, Anal. Biochem. 166, 90 (1987).
[39]
tRNA SPLICINGENDONUCLEASEFROMS. cerevisiae
473
EDTA, and 2.0 mg/ml proteinase K and incubated at 50° for 15 min. The products are mixed with 5/zl of 8 M urea, 20% sucrose, bromphenol blue, and xylene cyanol, heated to 65° for 5 rain, and separated by electrophoresis through a 10% polyacrylamide (acrylamide-bisacrylamide, 30: 1) gel containing 90 mM Tris-borate (pH 8.3), 2.5 mM EDTA, and 4 M urea) The 32p-labeled half-molecules are located by autoradiography and cut out of the gels, and the Cerenkov radiation is measured assuming a counting efficiency of 30%. A unit of activity is defined as the amount of enzyme needed to catalyze the cleavage of 1 x 10-12 mol of tRNA precursor.
Protein Determination Samples are precipitated with 5% trichloroacetic acid, 8,9 and the protein concentration is estimated by the method of Lowry et al.,10 using bovine serum albumin as the standard. Solutions contain 1% SDS to prevent interference by Triton X-100.
Electrophoresis Protein samples are precipitated in 1 ml of 5% trichloroacetic acid. After a 5-min centrifugation in a microcentrifuge, Tris base is added to the solution to a concentration of 0.3 M. Triton X-100 redissolves upon mixing, and precipitated protein is isolated by centrifugation for 10 min. The pellet is resuspended in 15/zl of sample buffer plus 2/zl of 2 M Tris base, heated for 3 min at 100°, and separated on a 5-15% polyacrylamide gradient gel according to Laemmli. H Separated proteins are visualized by silver staining) 2
Growth o f Yeast The protease-deficient S. cerevisiae strain 20B-12-1 (a-pep4-3, prcl, prbl, his) 5 is grown to a n A600 of 4-6 at 30° in 350 liters of 1% yeast extract, 2% peptone, and 2% glucose. The yeast are stored at - 7 0 ° until needed.
Purification Scheme Pellets and solutions are stored at - 2 0 °, and all steps are performed at 4° . s A. Bensadoun and D. Weinstein, Anal. Biochem. 70, 241 (1976). 9 p. R. Green, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 256, 11151 (1981). 10 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). it U. K. Laemmli, Nature (London) 227, 680 (1970). i2 W. Wray, T. Boulikas, V. P. Wray, and R. Hancock, Anal. Biochem. 118, 197 (1981).
474
PROCESSINGOF TRANSFERRNAs
[39]
TABLE I PURIFICATION OF YEAST tRNA SPLICING ENDONUCLEASEu
Purification step
Total activity (units)
Total protein (mg)
Specific activity (units/mg)
Purification (-fold)
Yield (%)
Triton X-100 extract Heparin-agarose 1 Heparin-agarose 2 Hydroxyapatite flow-through Hydroxyapatite Affi-Gel Blue tRNA elution
570 767 637 590 513 95 87
5,400 439 135 50 11.6 0.89 0.40 b
0.11 1.8 4.7 11.8 44.2 107 218
1 16 43 107 402 970 1,980
100 135 112 104 90 17 15
a The endonuclease was purified from 3 kg of yeast. Details of the steps employed are described in the text. b Protein was estimated by comparing volumes of the fraction loaded onto the SDSpolyacrylamide gel and the intensity of the stained protein bands between the Affi-Gel Blue and tRNA elution steps (see Fig. 2, lanes 7 and 8).
Preparation of Detergent Extract. Frozen cells are thawed in 2 volumes of 100 m M Tris-HCl (pH 8.0), 20 m M EDTA, 10% glycerol, 5 mM spermidine-HCl, 3 mM dithiothreitol, 1.0 M ammonium sulfate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells are lysed with glass beads (0.20-0.30 mm diameter; Glen Mills, Inc., Maywood, NJ) by passing them twice through a Dynomill chilled at - 1 0 °. The extract is centrifuged at 4,000 g for 10 min and the supernatant at 150,000 g for 2 hr. The membranes are homogenized into 2 volumes of lysis buffer using a Potter-Elvehjem homogenizer and centrifuged as above. The membranes are washed twice further by homogenizing into 20% glycerol, 5 mM 2mercaptoethanol, 25 m M Tris-HC1 (pH 8.0), 0.2 mM PMSF (buffer E), adding 0.02% Triton X-100, and stirring for 1 hr. The washed membranes are homogenized into 2 volumes of buffer E (20-40 mg/ml protein), Triton X-100 is added to 0.7%, and the mixture is stirred for 1 hr and centrifuged. This extract contains 5-50% of the extractable activity. The remaining activity is extracted as above using 0.9% Triton X-100 plus 0.1 M ammonium sulfate. Since activity is difficult to quantitate while membrane bound, the detergent extracts are defined to be 1-fold purified (Table I, Fig. 2, lane 2). The extracts may be stored indefinitely at - 2 0 °. Because of the low yield of endonuclease activity, we find it necessary to process kilogram quantities of yeast. To obtain higher yields of membranes, it is critical to centrifuge a full 2 hr. Still, much activity is found in the milky supernatant, perhaps in very small vesicles.
[39]
tRNA SPLICINGENDONUCLEASEFROMS. cerevisiae
475
Heparin-Agarose Chromatography. The extracts are made 2.5% in Triton X-100 by adding a 20% stock solution. The extract is applied to a 4 × 12 cm column of heparin-agarose that has been equilibrated with buffer C [25 m M Tris-HCl (pH 8.0), 10% glycerol, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100] containing 0.1 M ammonium sulfate. Bound protein is eluted at 150 ml/hr with a 2-liter gradient of 0.1-0.6 M ammonium sulfate in buffer C (Fig. 1A and Fig. 2, lane 3). Active fractions are combined, diluted 3-fold with buffer C, and applied to a 2 × 38 cm heparin-agarose column. Activity is eluted at 70 ml/hr with a l-liter gradient of 0.1-0.6 M ammonium sulfate (Fig. 1B and Fig. 2, lane 4). Hydroxyapatite Chromatography. The active heparin-agarose fractions are combined but cannot be dialyzed without extensive precipitation and loss of activity. They are applied to a 2 x 35 cm column of hydroxyapatite (BioGel HTP, Bio-Rad) equilibrated with buffer C containing 0.35 M ammonium sulfate. Most of the activity flows through the column (Fig. 2, lane 5). The flow-through is collected, diluted 3-fold with buffer C, and reapplied to the hydroxyapatite column equilibrated with buffer C. Activity is eluted at 70 ml/hr with a 1,100-ml gradient of 10% glycerol, 5 m M 2-mercaptoethanol, 0.5% Triton X-100, and 0-0.45 M potassium phosphate (pH 7.0) (Fig. 1C and Fig. 2, lane 6). Affi-Gel Blue Chromatography. The active hydroxyapatite fractions are combined and dialyzed against buffer C until the potassium phosphate concentration is less than 75 mM, then applied to a 1 x 16 cm column of Affi-Gel Blue (Bio-Rad). Activity is eluted at 30 ml/hr using a 1,100-ml gradient of buffer C containing 0-2.0 M NaC1 (Fig. 1D and Fig. 2, lane 7). The yield of activity is consistently low (10-20%). It can be increased by using a smaller column ( - 7 ml); however, this greatly decreases the efficiency in removing certain contaminants. tRNA Affinity Elution. Active fractions from the Affi-Gel Blue step are combined, dialyzed against buffer C, and applied to a 1 x 8 cm column of CM-Sepharose. The column is washed with 25 ml of buffer C containing 75 mM NaC1, and activity is eluted at 40 ml/hr with 100 ml of the same buffer plus 100 mg/ml yeast tRNA (Type X-S, Sigma, treated with proteinase K, phenol extracted, and ethanol precipitated) (Fig. 2, lane 8). Glycerol Gradients. The affinity eluant is concentrated by applying it to a 1-ml column of hydroxyapatite and step eluting with 10 ml of buffer C containing 0.4 M potassium phosphate (pH 7.0). A portion (300 /zl) is layered on an ll-ml, 10-16% glycerol gradient in buffer C plus 0.4 M potassium phosphate (pH 7.0). The gradients are centrifuged at 38,000 rpm (180,000 g) for 48 hr in an SW41 rotor. The purification is summarized in Table I and Fig. 2. The overall purification through the tRNA affinity column is about 2,000-fold over
476
PROCESSING OF TRANSFER R N A s A
/
v
0.6
x
×/× /
2.00
"7
[39]
0.80~
0.5 v
E LSO
0.60 ~
>I - 1.00 > I.,-
Z 0.40 ~
v
0.4. ~
c~ or) O.3 ~
h--
0.20
0.50
13_
0 I
Z 0.2 ' v
,
0.1
I
I
I
20
40
FRACTION
I
I
60
NUMBER
Bf
/x
4.oo
72.oot
tt 0.6 lo. o lo, o.eo "~"
o~
/, ~, ix
0
I
I0
20
FRACTION
30
OI
40
NUMBER
FIG. 1. Elution profiles. (A) Chromatography of Triton X-100 extract over heparinagarose. (B) Second chromatography over heparin-agarose. (C) Chromatography over hydroxyapatite. (D) Chromatography over Affi-Gel Blue. Active fractions combined for the next step are indicated by a bar in the figures.
[39]
tRNA SPLICING ENDONUCLEASE FROM S. cereoisiae
477
O.OeO? ]0.50
?4.o
1o.o io. o
/x x
~[~o
E
'w
1oo,,o~1o.~o~ z
x/
l
,
0
I0
T
13_
,
,
20 :50 40 FRACTION NUMBER
0_
]0
50
2.0
D X
4.0
0.04().~
1.6
0.03([
1.2
0.02( Z
0.8 :~
Q v
X
"~3.0
v
5
v
~ 2.0
P_
> I.~O
