- Email: [email protected]
Purification and properties of Escherichia coli methyl-deficient phenylalanine tRNA
382
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96404
P U R I F I C A T I O N AND P R O P E R T I E S
OF E S C H E R I C H I A
METHYL-DEFICIENT PHENYLALANINE
COLI
tRNA
NAOMI BIEZUNSKI*, D. GIVEON AND U. Z. LITTAUER Department o[ Biochemistry, Weizmann Institute o[ Science, Rehovot (Israel) (Received October 8th, I969)
SUMMARY M e t h i o n i n e - s t a r v e d t R N A was extracted from a "relaxed" methionine-requiring m u t a n t of Escherichia coli grown in a m e d i u m deficient in methionine. The m e t h y l deficient t R N A T M a n d n o r m a l m e t h y l a t e d t R N A phe species, found in u n f r a c t i o n a t e d m e t h i o n i n e - s t a r v e d t R N A , were separated a n d extensively purified employing chrom a t o g r a p h y on reversed-phase columns, b e n z o y l a t e d DEAE-cellulose a n d m e t h y l a t e d a l b u m i n silicic acid columns. The purified t R N A ph~ species were characterized b y their a m i n o acid accepting capacity a n d e x t e n t of m e t h y l a t i o n . Exposure of n o r m a l E. coli t R N A Ph~ to p H 2. 9 led to changes in its chromatographic p a t t e r n on m e t h y l a t e d a l b u m i n - s i l i c i c acid columns.
INTRODUCTION The s t u d y of the role of m e t h y l a t e d bases in t R N A has been made possible b y the f i n d i n g of MANDEL AND BOREK1,2 t h a t methyl-deficient t R N A is synthesized b y relaxed m e t h i o n i n e - r e q u i r i n g m u t a n t s of Escherichia coli d u r i n g m e t h i o n i n e starvation. t R N A isolated from such a m e t h i o n i n e - s t a r v e d E. coli culture is a m i x t u r e of methyl-deficient t R N A a n d m e t h y l a t e d n o r m a l t R N A . I n the case of m e t h i o n i n e starved t R N A Phe the methyl-deficient a n d n o r m a l species can be resolved b y methylated a l b u m i n - s i h c i c acid c o l u m n c h r o m a t o g r a p h y 3,4 c o u n t e r c u r r e n t d i s t r i b u t i o n s a n d reversed-phase c h r o m a t o g r a p h y s. I n order to s t u d y more closely the properties of methyl-deficient t R N A ~he species a n d compare t h e m to n o r m a l t R N A Phe we have developed procedures for their separation a n d extensive purification. Nomenclature: In this paper methioniue-starved tRNA refers to the tRNA extracted from a relaxed methionine-requiring mutant of E. coli (K12W6) grown in a medium deficient in methionine. Methyl-deficient tRNA refers only to those species which contain few or no methyl groups. Methionine-starved tRNA is a mixture of methyl-deficient tRNA and methylated normal tRNA. tRNAPhe (NN), methylated normal tRNAPhe synthesized under normal growth condition; tRNAPhe (DS), methyl-deficient tRNAPhe isolated from methionine-starved cells; tRNAPhe (NS), normal tRNAPhe isolated from methionine-starved cells; tRNAPhe (DS+NS) methionine-starved tRNAPhe containing a mixture of tRNAPhe (DS) and tRNAPhe (NS). * Present address: Department of Virology, Hadassah Medical School, The Hebrew University, Jerusalem, Israel. Biochim. Biophys. Acta, 199 (197o) 382-393
METHYL-DEFICIENTtRNA Phe
383
MATERIALS AND METHODS
Growth o/ E. coli cells and preparation o/tRNA E. coli K12W6 RC tel m e t - was obtained from Dr. Esther Lederberg. Normal cultures were grown in minimal medium 7 containing o.oi M potassium phosphate buffer (pH 7.4) supplemented with 50/~g/ml of L-methionine at 37 ° with forced aeration. Cells were harvested in the middle of the logarithmic growth phase and the tRNA isolated immediately according to a previously described procedure s, except that the final dialysis step was omitted. The t R N A was then incubated at 37 ° in Tris-HC1 buffer (pH 8.8, 0.2 M) for 3 h. Methionine-starved cultures were grown as follows: a starter culture supplemented with 8/~g/ml of L-methionine was grown overnight with slow shaking. 600 ml of this culture were inoculated into 12 1 of medium supplemented with 4/~g/ml of L-methionine. The culture was grown at 37 ° with forced aeration. After growth had stopped, the culture was further incubated with continued aeration for an additional 3.5 h. All the tRNA preparations were routinely assayed for the presence of nuclease contamination s. Aliquots of the t R N A solution were diluted with I vol. of autoclaved 0.2 M Tris-HC1 buffer (pH 8.8) and held at 37 ° over chloroform for 25 h. The t R N A was then isolated by ethanol precipitation and assayed for its capacity to accept phenylalanine, tRNA preparations showing more than 15 % decrease in their acceptor activity were discarded.
Phenylalanyl-tRNA synthetase Normal E. coli K12W6 cells (2oo g) were suspended in 6oo ml of o.oi M TrisHC1 buffer (pH 7.8) containing o.oi M MgC12 and I/~g/ml of deoxyribonuclease at 4 °. The suspension was homogenized in a Mantan-Gaulin homogenizer (Everett, Mass.) by recycling it 3 times at 4000 lb-inch -2. The lysate was centrifuged for IO rain at 15 ooo ×g and the precipitate discarded. The supernatant fluid was centrifuged at lO5 ooo ×g for 2 h. The lO5 ooo × g supernatant was stirred and o.I vol. of io % streptomycin sulfate solution (freshly prepared and neutralized to pH 7.0) was added. After 30 min of stirring, precipitated material was removed by a Io-min centrifugation at 15 ooo ×g, and the supernatant fraction adjusted to pH 8.0 with 0.2 M NaOH. (NHa)~S Q fractionation was next carried out, and the 30-39 % saturation cut recovered by centrifugation. This protein fraction was dissolved in 60 ml of 0.02 M Tris-HC1 buffer (pH 7.4). To this solution were added: 0.2 vol. of o.I M ATP, 0.2 vol. of o.I M L-phenylalanine, 0.04 vol. of 0.5 M MgC12, o.14 vol. of 0.05 M K F and 0.06 vol. of 0.5 M of K2HPO 4. This mixture was incubated at 55 ° for 45 min, and denatured protein was removed by chilling and centrifugation. 1.6 vol. of saturated (NH4)2SO 4 solution was now added to the supernatant fraction. The precipitate was collected by centrifugation, dissolved in 12 ml of 0.02 M Tris-HC1 buffer (pH 7-4) and dialyzed overnight against the same buffer. Traces of residual nuclease were removed by chromatography through a 2 cm × 20 cm DEAE-cellulose column 9. The column was equilibrated with initial buffer consisting of 0.02 M potassium phosphate l~uffer (pH 7-5), 0.02 M 2-mercaptoethanol and o.ooi M MgC12. The extract was applied, and the column was washed with 500 ml of the initial buffer. The enzyme was eluted with a buffer containing 0.25 M potassium phosphate (pH 6.5), 0.02 M 2-mercaptoethanol, and o.ooi M MgC12. Peak activity fractions were pooled, dialyzed against Biochim. Biophys. Acta, 199 (197o) 382-393
384
~. BIEZUNSKI et al.
o.oi M Tris-HC1 buffer (pH 7.4) and dithiothreitol was then added to a final concentration of 0.25 mg/ml. The enzyme solution was distributed into small aliquots and stored frozen at --15 °. Dialysis bags were boiled twice in 5 % Na2CO3, rinsed with water, then boiled in 0.05 M E D T A (pH 8.0) and stored in o.I mM EDTA.
Amino acid acceptance assay For assay of Phe-tRNA formation, the reaction mixture (o.o5 ml) contained: 2. 7 #moles of Tris-HC1 buffer (pH 7.8); o.I/,mole of ATP; o.I/*mole of GSH (neutralized); I.O/*mole of MgC12; 15o/*/*moles of El*C~phenylalanine (4" lO5 counts/min per m#mole)' 200 ##moles of each of the 19 other 14C-labeled amino acids; O.l-O.2 A26o ms units of unfractionated t R N A or about o.oi A280 m # units of purified t R N A Phe and 12/~g of enzyme protein. After a Io-min incubation at 37 °, the assay tubes were mixed with 0.2 mg of serum albumin, and 3 ml of 5 % trichloroacetic acid was added. After IO rain at 4 ° the precipitates were collected onto membrane filters, washed twice with 5-ml portions of 5 ~o trichloroacetic acid, dried, placed in IO ml of toluene scintillation fluid and radioactivity was counted in a Packard Tri-Carb scintillation spectrometer. For assay of amino acid acceptance of tRNA, a mixed aminoacyl-tRNA synthetase was prepared from E. coli A-I 9 cells according to the procedure of MOENCH AND BERGs up to and including the DEAE-cellulose step. The reaction mixture (0.05 ml) contained: Tris-HC1 buffer, ATP, GSH and MgC12 as detailed above for phenylalanine acceptance assay, and 0.2/~C of a mixture of 15 14C-labeled L-amino acids mixed together with IO re#moles of unlabeled L-phenylalanine; 0.03-0.2 A260ms units of t R N A and 53 #g of enzyme protein. The mixture was incubated for io min at 37 °. The subsequent steps were the same as for the assay of Phe-tRNA. The 14Clabeled amino acid mixture was purchased from New England Nuclear Corp. and contained a mixture of 15 L-amino acids at approx. 40 mC/matom of carbon. I mC of this mixture contained: alanine (80 #C), arginine (7° #C), aspartic acid (80/~C), glutamic acid (125 #C), glycine (4°/~C), histidine (15/~C), isoleucine (5 ° #C), leucine (14o/~C), lysine (60/~C), phenylalanine (80 ~C), proline (50/~C), serine (4° #C), threonine (50/~C), tyrosine (40/zC) and valine (80/~C). The concentration of t R N A was estimated by measuring the absorption at 260 m/~. I m g of t R N A was found to elicit an A260 ms of 30 units in o.oi M NaOH (ref. IO) or, alternatively an A260 ms of 25 units in 0.5 M NaC1. The values measured in N a O H were found to be more accurate, and less variable than those obtained in NaC1, consequently the NaOH values were routinely employed for calculation of the amino acid acceptor capacity of tRNA. Whenever the results are expressed in A260 ms units they were measured in 0.5 M NaC1. A ssay method/or tRNA-methylase E. coli K12W6 cells were grown in a minimal medium 7 containing o.oi M potassium phosphate buffer (pH 7.4) and 0.2 % casamino acids at 37 °. The cells were harvested in the early exponential growth phase, washed once with 0.025 M TrisHC1 buffer (pH 8.0) and frozen until use. A mixed tRNA-methylase was made b y grinding frozen cells with 2.5 times wet weight of alumina and extracting the paste with 2. 4 times wet weight of buffer (o.oi M MgC12; o.oi M Tris-HC1 buffer (pH 7.8); Biochim. Biophys. Acta, 199 (i97o) 382-393
METHYL-DEFICIENTt R N A Phe
385
o.oo2 M GSH (neutralized); I/~g/ml deoxyribonuclease; and IO °/o glycerol) at 4 °. The suspension was centrifuged for 15 min at 15 ooo x g and the supernatant was further centrifuged for 2 h at 15o ooo xg. The supernatant was stirred and 0.087 vol. of neutralized IO % streptomycin sulfate solution was added. After 20 min of stirring, precipitated material was removed b y a Io-min centrifugation at 20 ooo xg. The supernatant (9 ml) was applied onto a Sephadex G-5o column (3 cm x 3 o cm) equilibrated with o.oi M Tris-HC1 buffer (pH 7.4) containing 0.002 M GSH (neutralized) and io % glycerol. The enzyme was eluted with the same buffer, peak activity fractions were pooled, GSH and glycerol were added to a final concentration of o.o15 M and 30 % respectively, and stored at - - 1 5 °. For assay of t R N A methylation the reaction mixture (o.15 ml) contained: 3 /,moles of Tris-HC1 buffer (pH 9.0; the final p H is 8.8); 0.57 #mole of MgC1d o.17 /*mole of GSH (neutralized); 13. 5 m#moles of E14C]methyl-S-adenosyl-L-methionine (6.6. IO4 counts/rain per m/*mole); 20/,moles of ammonium acetate; 1.5- 5/*g of t R N A and 250/*g of enzyme protein. After a 6o-min incubation period at 37 °, the assay tubes were mixed with 0.2 mg of serum albumin, and 3 ml of cold 5 % trichloroacetic acid was added. After io min at 4 ° the precipitates were collected onto membrane filters, washed twice with 5-ml portions of 5 % trichloroacetic acid and 5 times with 5 % trichloroacetic acid solution containing o.oi M sodium pyrophosphate. The membrane were then dried and counted. C o l u m n chromatography
Reversed-phase column chromatography was carried out as described b y KELMERS et al. 11 and KELMERSTM. Two parts of Chromosorb W were suspended in one part of 4 % dimethyldilaurylammonium chloride in isoamyl acetate and poured into a jacketed glass column (2.5 cm x 19o cm containing 0.45 M NaC1, o.oi M MgC12 and o.oi M sodium acetate buffer (pH 4-5) saturated with isoamyl acetate at 37 ° (initial buffer). The t R N A was dissolved in IO ml of initial buffer and applied to the column. Linear gradient elution was carried out at 37 ° using 1.8 1 of initial buffer and 1.8 1 of a solution containing 0.75 M NaC1, o.oi M MgC12 and o.oi M sodium acetate buffer (pH 4-5) saturated with isoamyl acetate. The column eluate was collected at a flow rate of 60 ml/h, and o.o4-ml aliquots were removed and assayed for amino acid acceptance. Benzoylated diethylaminoethyl-cellulose-column chromatography was carried out as described by GILLAM et al. 13. The benzoylated diethylaminoethyl-cellulose column (0. 9 cm x 95 cm) was washed with a solution consisting of 2 M NaC1 and 25 % ethanol and then equilibrated with a solution 14 containing 0.4 M NaC1 and 0.05 M sodium acetate buffer (pH 5.0) (initial buffer). The t R N A solution was applied to the column then washed with 7 ° ml of initial buffer and linear gradient elution was carried out as indicated. Methylated albumin-silicic acid columns (2 c m x 12 cm) were prepared as previously described 4. To enhance the methylated albumin-silicic acid-column resolution the labeled Phe-tRNA samples were mixed together with 4 mg of uncharged normal tRNA. The t R N A solution (i.o ml) was diluted with 9.0 ml of 0.8 M NaC1 solution containing 0.05 M sodium acetate buffer (pH 5.5) (initial buffer) and applied to the column. The column was washed with 50 ml of initial buffer and the t R N A was then eluted at 16 ° with a linear gradient containing 200 ml of initial buffer and 200 ml of Biochim. Biophys. Acta, 199 (197o) 382-393
386
N. BIEZUNSKI et al.
i.o M NaC1 solution containing o.o5 M sodium acetate buffer (pH 5.5). Fractions of 3.o ml each were collected at a flow rate of 3o ml/h. Each receiving tube contained o.2 ml of I M sodium acetate buffer (pH 4.5). Assay of acid insoluble radioactivity was carried out as previously described 4. Throughout the purification of t R N A care was taken not to introduce nucleases b y using sterile buffer solutions and glassware. Glass columns were washed with o.I M NaOH, o.I M HC1 and water before use. MATERIALS
Dimethyldilaurylammonium chloride (Aliquat 2o4) was a gift from the Chemical Division of General Mills, Ill., and was recrystallized 3 times from acetone at --2o °. Chromosorb W (acid-washed, dimethyldichlorosilane-treated, lOO-2Oo mesh size) was obtained from Johns Manville. Benzoylated diethylaminoethyl-cellulose ( 5 o - I o o m e s h ) and L@4Clphenylalanine (255 mC/mmole) were purchased from Schwartz BioResearch. [14C]Methyl-S-adeno.,yl-L-methionine (36 mC/mmole) was obtained from Tracerlab. Membrane filters (MF-5o) with pore size of 0.6 # were purchased from Membranfiltergesellschaft, G6ttir.gen. Levigated alumina was obtained from Norton Co. and glycerol from Matheson, Coleman and Bell. RESULTS
Puri/ication o/ normal and methyl-deficient t R N A Phe Reversed-phase chromatography of methionine-starved t R N A resolves two peaks of t R N A Phe (Fig. Ib). The first peak corresponds to methyl-deficient tRNA Phe
0.4 ~
20.0
tRNA phi (NN) I0.
~000
//
ZL
~
o
=
o
tO00
1.0 E
o
I
ea
J
,
'
" -
l
o.e =-
....
zo.o
7ili
2.0
1.0
NA ~ (DS)
0.4 ~
,3000
!10000
~ " =
(NS)
-
z;O0
BOO
3
:oooo
1200
Effluent
1600
2000
.5000
2400
volume ( m l )
F i g . i. R e v e r s e d - p h a s e c o l u m n c h r o m a t o g r a p h y of n o r m a l a n d m e t h i o n i n e - s t a r v e d t R N A . a. I7O m g of n o r m a l t R N A w e r e a p p l i e d t o t h e c o l u m n a n d 9 - m l f r a c t i o n s w e r e c o l l e c t e d , b. 1 8 o m g o f methionine-starved tRNA were applied to the column and 8-ml fractions were collected. Q-O, phenylalanine acceptance (o.o4-ml aliquots); - - , A 260 my.
Biochim. Biophys. Acta, 199 (197 o) 3 8 2 3 9 3
MLTHYL-DEFICIENT t R N A Ph~
387
(DS), while the second peak represents normal t R N A vhe (NS) (see below). This separation is similar to that obtained b y SHUGARTet al. e. I t was also consistently observed that the methyl-deficient t R N A Phe (DS) had a tendency to separate into two components, these were resolved on methylated albumin-silicic acid-column chromatog:aphy (see below). The elution profile of methylated normal tRNA, isolated from log-phase cells grown in the presence of an excess of methionine, is shown in Fig. Ia. The major peak corresponds to normal t R N A vh~ (NN), while a trace of methyl-deficient t R N A Phe is also evident. The peak fractions containing t R N A T M were pooled, concentrated and freed of salts by ultrafiltration. The concentrated t R N A Phe fractions were then subjected .
L6
(0)]
l 0e ~,
112°°
0O8
1
400
0.04
1"6i 0.8
~ - - "~ - --
0,8 u:
16000
0.6 ~
12000
o.4
8o00
0.2
4000 u
16
~
(c:
~0~ o.oe
0.04 I'L
500 t 20
40
60
80 Tube
I00
120 140 160
number
Fig. 2. Benzoylated diethylaminoethyl-cellulose column c h r o m a t o g r a p h y of enriched tRI~APhe fractions, a. tRNAPhe (NN) fractions (between 1575 to 1638 ml, Fig. Ia) were pooled and concentrated b y ultrafiltration t h r o u g h a U M - I o m e m b r a n e (Diaflo ultrafiltration cell Amicon Corp., Lexington, Mass.) to a volume of 4 ml. The c o n c e n t r a t e d solution was diluted with 4 ° ml of w a t e r and t h e n c o n c e n t r a t e d again to a b o u t 2.0 ml. The t R N A solution was applied to the column and w a s h e d w i t h 7 ° ml of o. 4 1V[ NaCI solution containing 0.o 5 M sodium acetate buffer {pH 5.0) (initial buffer). The linear gradient elution was carried o u t at 4 ° using 2oo ml of initial buffer and 200 ml of I . t M NaC1 solution containing o.o 5 M s o d i u m acetate buffer (pH 5.0). This was followed b y a second gradient containing 5 ° ml of i . I M NaC1 and 5 ° ml of 1. 5 M NaC1 containing o.o 5 M s o d i u m acetate buffer (pH 5.0). 4-ml fractions were collected at a flow rate of 36 ml/h. F r a c t i o n s No. 9 4 - t o 2 were pooled, concentrated b y ultrafiltration to a b o u t 4 ml, t h e n diluted w i t h 40o ml of w a t e r and c o n c e n t r a t e d again to a b o u t 2.0 ml. b. The pooled and concentrated tRNAPhe (DS) fractions (between 11o 4 to 12o8 ml, Fig. lb) were applied to the c o l u m n and eluted as described u n d e r a. 4-ml fractions were collected at a flow rate of 48 ml/h. Fractions No. 84-95 were pooled, concentrated and freed of salt as described above, c. The pooled and concentrated tRNAPhe (NS) fractions (between 1688 to 1792 ml, Fig. Ib) were applied to the c o l u m n and eluted as described u n d e r a except t h a t the second eluting gradient consisted of ioo ml of I . I M NaC1 and IOO ml of 1.5 M NaC1 containing 0.o5 M s o d i u m acetate buffer (pH 5.o). 4-ml fractions were collected at a flow r a t e of 48 ml/h. Fractions No. 94-115 were pooled, c o n c e n t r a t e d and freed of salt as described above. Q - Q , phenylalanine acceptance (o.o4-ml aliquots); , A260 me*.
Biochim. Biophys. Acta, 199 (t97 o) 382-393
N. BIEZUNSKI et al.
388
to benzoylated diethylaminoethyl-cellulose chromatography (Fig. 2). In the case of t R N A vhe (NN), and possibly t R N A vhe (NS), the remain tRNA TM peak was found to be followed b y a smaller shoulder. Table I shows the extent of amino acid acceptance of the various t R N A vhe fractions. The combination of reversed-phase column chromatography and benzoylated diethylaminoethyl-cellulose-column chromatography yielded highly purified t R N A vh~ preparations exhibiting a phenylalanine acceptance value of 30-36 m/~moles per mg of t R N A (or 12oo-144o #/zmoles per A2s0 ma unit). Further attesting to the high degree of purification we have achieved is the decidedly reduced ability of all preparations to accept amino acids.
TABLE I PURIFICATION
OF N O R M A L A N D
METHIONINE-STARVED tl~NA phe
Sample
Initial normal tRNAPhe (NN) Reversed phase of tRNAPhe (NN) Benzoylated diethylaminoethylcellulose of tRNAPhe (NN) Initial methionine-starved tRNAPhe (DS+NS) Reversed phase of tRNAPhe (DS) Benzoylated diethylaminoethylcellulose of tRNAPhe (DS) Reversed phase of tRNAPhe (NS) Benzoylated diethylaminoethylcellulose of tRNAPhe (NS)
Methyl groups incorporated (mttmoles/mg RNA )
A mino acids incorporated Phenylalanine (mlzmoles/mg RNA )
Amino acid mixture* (counts/rain per i~g R N A )
o.41 1.44
1.25 14. 5
388o 191o
0.77
30.2
720
45.9 49.7
1.47 21.6
442o 154o
6o. 4 18.9
35.9 2o. 4
48o 148o
lO. 7
29.7
465
* Assayed with crude aminoacyl-tRNA synthetase and a mixture of 15 14C-labeled L-amino acids mixed together with unlabeled L-phenylalanine.
The methyl group accepting capacities of the t R N A Phe fractions were assayed with a mixture of crude tRNA-methylase from E. coli (Table I). When normal t R N A Ph~ (NN) was used as methyl acceptor very little methylation occurred; on the other hand with methyl-deficient t R N A Phe (DS) a substantial amount of methyl groups were incorporated. The normal t R N A Phe (NS) fraction isolated from the unfractionated methionine-starved t R N A showed a significant ability to accept methyl groups and seems still to be contaminated with some species of methyl-deficient t R N A other than t R N A TM.
Methylated albumin-silicic acid-column chromatography of the purified t R N A Phe fractions The identity of the various t R N A Phe fractions was verified b y methylated albumin-silicic acid-column chromatography 4. Fig. 3 a shows the elution profile of unfractionated [14ClPhe-tRNA (NN) and unfractionated methionine-starved [sH]Phe Biochim. Biophys. Acta, 199 (197 o) 382 393
389
METHYL-DEFICIENT t l ~ N A Phe I
I
I
I
I
1
{o)
U nfroclionoted
[~'H]phl"IRNA{0S*NS) L6
--
/
1,2 0.8
I
I
I
(b)
UnfractionQted [3H]P.e-tRNA (NN)
3H
14C
I
1800
6 0 0 0 I'-
~(NN)
3H
~/ Pu,ili,d
4000 t4000
12ooo !2000
,oooooVtI
Oa
Unfroclionoted
(c)
~.3H]Phe-tRNA(05÷N$) Purified
12
E
"~-
I I I I UnfractioneO
]
I (d)
[3HIPhe_l RNAtOS+NS ) 3H I'4Cfi-~ ,~! :! [:Ooi]l~i ,~i'~dN A(NS) 3000
, :i.
14C
6000
.
,~ '.
:
3H
14c
~.000
/
6000
i! 2000
0.8
,ooo
0.4
20
40
60
80
ooo
I00
2000
20 Tube
40
60
80
I00
number
Fig. 3. Methylated albumin silicic acid-column chromatography of P h e - t R N A fractions, a. O - O , 12o ooo counts/rain of unfractionated normal [14C]Phe-tRNA (NN); O - - -O, ioo ooo counts/min of unfractionated methionine-starved ~ H ] P h e - t R N A (DS + N S ) . b. O - Q , 7 ° ooo counts/rain of purified [14C]Phe-tRNA (NN) fractions (No. 94-1o2, Fig. 2a); O - - - O , IOO ooo counts/rain of unfractionated normal F3H]Phe-tRNA (NN). c. 0 - 0 , 220 ooo counts/min of purified []4C]Phet R N A (DS) fractions (No. 84-95, Fig. 2b); O - - - O , I7OOOOCounts/min of unfractionated methionine-starved [3H]Phe-tR~A ( D S + N S ) . d. Q - O , ioo ooo counts/min of purified [14CJPhet R N A (NS) fractious (No. 9 4 - i I ~ , Fig. 2c); O - - - O , 225 ooocounts/min of unfractionated methionine-starved [~H]Phe-tRNA ( D S + N S ) . All labeled l~he-tRNA samples were mixed together with 4 mg of uncharged normal t R N A (unlabeled); - - , A 26~ mr*.
tRNA (DS+NS) on a methylated albumin-silicic acid column. The methioninestarved [~H]Phe-tRNA resolved into three main peaks. The first two peaks eluting from the methylated albumin-silicic acid column correspond to methyl-deficient Phe-tRNA (Peaks a and b in Fig. 2, ref. 4) while the last peak corresponds to normal Phe-tRNA (Peak d mixed together with Peak c in Fig. 2, ref, 4). In some experiments an additional peak was observed (Peak c in ref. 4) which preceded the major peak of normal Phe-tRNA. However, its resolution was variable and depended on the particular batch of methionine-starved tRNA as well as on the sample of methylated albumin-silicic acid used for the column chromatography. The assignments of the methylated albumin-silicic acid-column peaks were verified by rechromatography on methylated albumin-kieselguhr columns, on which the nature of the peaks had already been established in an earlier communication 1~. Fig. 3b shows that purified [14C]Phe-tRNA (NN) and unfractionated [3H]Phe-tRNA (NN) coeluted from the methylated albumin-silicic acid column. Purified methyl-deficient EI*C]Phe-tRNA (DS) fraction resolved into two species (Fig. 3c) that appear to coincide with the first two peaks of methionine-starved [~H]Phe-tRNA (DS+NS). The behavior of purified [14C]Phe-tRNA (NS) upon methylated albuminsilicic acid-column chromatography is depicted in Fig. 3. The results indicate that Biochim. Biophys. Acta, 199 (197 o) 382-393
390
N. BIEZUNSKI et al.
this fraction has similar chromatographic properties to the last peak of unfractionated methionine-starved Phe-tRNA as well as to Phe-tRNA isolated from E . coli cells grown under normal conditions. Since reversed-phase chromatography necessitated prolonged exposure of tRNA at pH 4.5 it was important to examine whether this treatment had any effect on this tRNA. Unfractionated methionine-starved tRNA was therefore incubated in the presence of the terminal buffer (0.75 M NaC1, o.oi M MgC12 and o.oi M sodium acetate buffer (pH 4.5) saturated with isoamyl acetate) for a period of 4 days at 37 °. The tRNA was then isolated by ethanol precipitation and its ability to accept phenylalanine was assayed. Only a slight decrease of activity was observed (1.24 vs. 1.51 m/zmoles of phenylalanine per mg RNA for the incubated and unincubated preparations respectively). Methylated albumin-silicic acid-column chromatography of the incubated E14C~Phe-tRNA together with the unincubated I~HIPhe-tRNA showed that both preparations had the same elution profile. Thus, by the methylated albumin-silicic acid-column criteria no adverse effects have occurred during the incubation of the tRNA at pH 4.5. It was, however, noted that exposure of tRNA to a lower pH did affect both its phenylalanine acceptor ability and its chromatographic properties on methylated albumin-silicic acid columns. Incubation of unfractionated normal tRNA at pH 2. 9 for 2 h at 37 ° according to the procedure of THIEBE AND ZACHAU16 resulted in a significant loss of activity (0.50 vs. 0.86 m#moles phenylalanine per mg RNA for the incubated and unincubated preparations respectively). In addition, methylated albumin-silicic acid-column chromatography revealed an altered elution profile for the pH-2. 9 treated Phe-tRNA. I
I
I
I
I
i /['4c]Phe-t~NA
"C
pH 2.9 treoted
000
i/i
~_.
~4000
I
1
.°° Phe-tRNA
0
12
I
600
0.8
400
Z400
E
-~16oo ~ /
20
40
60
80 Tube
I00
120
140
160
number
Fig. 4" Methylated albumin-silicic acid-column c h r o m a t o g r a p h y of pH-2.9 treated n o r m a l P h e - t R N A . 2 ml of u n f r a c t i o n a t e d n o r m a l t R N A (5 mg) was dialyzed overnight against w a t e r at 4% a d j u s t e d to p H 2.9 with a m m o n i u m f o r m a t e buffer (final concn, o.i M) and incubated at 37 ° for 2 h. The t R N A solution was a d j u s t e d to p H 7.4 with I M Tris-HCl buffer (pH 8.8) and an aliquot was r e m o v e d and charged with Ex4C]phenylalanine. The pH-2.9 t r e a t e d CxIC]Phe-tRNA (27 5oo c o u u t s / m i n , ( D - G ) was mixed t o g e t h e r with u n t r e a t e d normal [ 3 H ] P h e - t R N A (60 ooo c o u n t s / m i n , O - O ) a n d 8 m g of carrier uncharged n o r m a l t R N A and applied to a m e t h y l a t e d albumin-silicic acid column. - - -, A 2n3 mu.
Biochim. Biophys. Acta, 199 (I97 o) 382-393
METHYL-DEFICIENT tRNAph~
391
Fig. 4 shows that following p H 2. 9 treatment two additional peaks of Phet R N A appear that are more retarded on the methylated albumin-silicic acid column than the main, normal Phe-tRNA component.
DISCUSSION
The ability of methyl-deficient t R N A to accept amino acids was studied in several laboratories. Early experiments 17-23 demonstrated that unfractionated methionine-starved t R N A preparations (consisting of a mixture of methyl-deficient and normal methylated tRNA) incorporated several amino acids to the same extent as fully methylated normal tRNA. In contrast to the above studies SHUGART et al. ~a reported that the extent of incorporation of four amino acids, namely phenylalanine, leucine, tyrosine and histidine, was decreased in unfractionated methionine-starved t R N A preparations. In addition, this group assayed the phenylalanine-accepting capacity of normal and methyl-deficient t R N A vhe separated by reversed-phase column chromatography. They found s that the total amount of phenylalanine accepted as well as the initial velocities oi phenylalanine incorporation correlate with the degree of undermethylation. In the present studies we have compared the phenylalanine accepting capacity of unfractionated normal and methionine-starved tRNA. Though the specific activities varied from one preparation to another, most of our unfractionated methioninestarved preparations showed a somewhat higher activity (lO-25 %) than the normal preparation. In none of the m a n y cases examined had the methionine-starved t R N A a lower activity than the normal tRNA. Table I shows that the methyl-deficient t R N A vhe species can be purified extensively. The purification that we have achieved (35.9 m#moles per nag RNA) would indicate that the methyl-deficient t R N A vhe is almost pure. Thus, under the condition of our assay system there is no indication t h a t the extent of phenylalanine acceptance of the methyl-deficient t R N A vhe is lower than that of the normal t R N A vh" species. It should be stressed that the success of the purification procedure was found to depend on the availability of t R N A preparations that are free of nuclease contamination (see METHODS), it also depended on the use of sterile buffer solutions and glassware. It was shown in the present studies that prolonged exposure of t R N A vhe in the pH-4.5 buffer, used for the reversed-phase chromatography, did not affect its phenylalanine accepting capacity or its chromatographic behavior on methylated albumin-silicic acid columns. As shown in Fig. 3, the purified Phe-tRNA (NN), Phe-tRNA (DS) and Phe-tRNA (NS) coeluted with the corresponding components of the unfractionated tRNA. Thus, b y the methylated albumin-silicic acid-column criteria no adverse effects have occurred during the purification of the t R N A vh" fractions. By contrast, is was found that exposure of t R N A vhe to a lower p H value, namely p H 2.9, does reduce its activity as well as alters its elution properties from methylated albumin-silicic acid columns. The pH-2.9 treatment was found by THIEBE AND ZACHAUle to excise from yeast t R N A phe the base Y+ which is located right next to the anticodon 25. The effect of the pH-2.9 treatment on E . coli t R N A vh*, however, is not due to excision of the base Y+ since the latter is absent from this t R N A species l°,zn. The methylated albumin-silicic acid elution profile of pH-2.9-treated Biochim. Biophys. Acta, I99 (I97o) 382-393
392
N. BIEZUNSKI et al.
tRNA Phe also does not correlate with the change in the methylated albumin-kieselguhr-column chromatographic profile of aged, normal Phe-tRNA preparations 15,3. In the latter case storage of frozen Phe-tRNA preparations resulted in the appearance of a new peak eluting from the methylated albumin-kieselguhr column at a lower salt concentration than the major Phe-tRNA peak. Whether the pH 2. 9 treatment affects the secondary structure of tRNA Phe or one of its minor bases remains to be determined. The inclusion of glycerol throughout the isolation procedure of the tRNAmethylase, was found to considerably enhance its activity. Thus, the extent of methylation of methionine-starved tRNA was increased by 2-2.5-fold. It was observed that about 50 % of the expected methyl acceptor sites of purified methyl-deficient tRNA Phe (DS) were methylated (60 vs. 113 m/~moles of methyl groups per mg RNA). These results may indicate that the purified methyl-deficient tRNA Ph~ species still contains a significant amount of methyl groups or that our crude tRNA-methylase was unable to methylate all the available acceptor sites. It should also be recalled that the methyl-deficient tRNA Pae (DS) can be resolved by methylated albuminsilicic acid-column chromatography into two components (Fig. 3). These may differ in their primary sequence or their degree of methylation. Another possibility is that these two methyl-deficient tRNA Ph~ components differ in the degree of oxidation of their thiolated bases or tile extent of covalent bond formation a7 between the 4-thiouridine in Position 8 and the cytidine residue in Position 13 resulting from the influence of ultraviolet light on methyl-deficient tRNA Ph*.
ACKNOWLEDGMENT
This research was supported, in part, by agreement Institutes of Health, U.S. Public Health Service.
No. 455114
of the National
REFERENCES I L. R. MANDEL AND E. BOREK, Biochem. Biophys. Res. Commun., 4 (1961) 14. 2 L. R. MANDEL AND E. BOREK, Biochemistry, 2 (1963) 560. 3 U. Z. LITTAUER, M. REVEL AND R. STERN, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 5Ol. 4 R. STERN AND U. Z. LITTAtlER, Biochemistry, 7 (1968) 3469 . 5 E. FLEISSNER, Biochemistry, 6 (1967) 621. 6 L. SHUGART, G. D. ~NTOVELLIAND M. P. STULBERG, Biochim. Biophys. Acta, 157 (1968) 83. 7 A. D. HERSHEY AND M. CHASE, J. Gen. Physiol., 36 (1952) 39. 8 U. Z. LITTAUER, S. A. YANKOFSKY, A. NOVOGRODSKY, H. BURSZTY'N, Y. GALENTER AND E. KATCHALSKI, Biochim. Biophys. Acta, 195 (1969) 29. 9 K. H. MUENCH AND 12). BERG, in G. L. CANTONI AND D. R. DAVIES, Procedures in Nucleic Acid Research, Harper and Row, New York, 1966, p. 375. IO P. BERG, F. H. BERGMANN,E. J. OFENGAND AND 1~I. DIECKMANN, J. Biol. Chem., 236 (1961) 1726. I I A. D. KELMERS, G. D. NOVELLI AND M. P. STULBERG, J. Biol. Chem., 240 (t965) 3979. 12 A. D. KELMERS, [. Biol. Chem., 241 (1966) 354 o. 13 I. GILLAM, S. 1V[ILLWARD, D. BLEW, M. VON TIGERSTROM, E. WIMMER AND G. M. TENER, Biochemistry, 6 (1967) 3043 . 14 K. L. RoY AND D. S6LL, Biochim. Biophys. Acta, 161 (1968) 572. 15 M. }{EVEL AND U. Z. LITTAUER, Biochem. Biophys. Res. Commun., 20 (1965) 187. 16 JR. THIEBE AND H. G. ZACHAU,European J. Biochem., 5 (1968) 546. 17 F. C. NEIDHARDT AND L. EIDLIC, Biochim. Biophys. Acta, 68 (1963) 380. 18 J. L. STARR, Biochem. Biophys. Res. Commun., io (1963) 181.
Bio=him. Biophys. Acta, 199 (197 o) 382-393
METHYL-DEFICIENT t R N A Ph"
393
19 I. SVENSSON, H. G. BOMAN, K. G. ERIKSSON AND K. KJELLIN, J. Mol. Biol., 7 (1963) 254. 20 U. Z. LITTAUER, K. H. MUENCH, P. BERG, W. GILBERT AND P. F. SPAHR, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 157. 21 A. PETERKOFSKY, C. JESENSKY, A. BANK AND A. H. MEHLER, J. Biol. Chem., 239 (1964) 2918. 22 U. Z. LITTAUER, Proc. 6th Intern. Congr. Biochem., 32 (1964) i i . 23 U. Z. LITTAUER AND R. MILBAUER, in Syrup. on R N A Structure and Function, Proc. 2nd Meeting Federation European Biochem. Socs., Vol. 4, P e r g a m o n , London, 1965, p. 9. 24 L. SHUGART, B. H. CHASTAIN, G. D. NOVELLI AND M. P. STULBERG, Biochem. Biophys. Res. Commun., 31 (1968) 404 . 25 W. L. RAJBHANDARY, S. H. CHANG, A. STUART, R. D. FAULKNER, t~. IV[. HOSKINSON AND H. G. KHORANA, Proc. Natl. Acad. Sci. U.S., 57 (1967) 75126 B. G. BARRELL AND F. GANGER, F E B S Letters, 3 (1969) 275. 27 M. YANIV, A. FAVRE AND B. G. BARRELL, Nature, in the press.
Bioehim. Biophys. Acta, 199 (197 o) 382-393
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96404
P U R I F I C A T I O N AND P R O P E R T I E S
OF E S C H E R I C H I A
METHYL-DEFICIENT PHENYLALANINE
COLI
tRNA
NAOMI BIEZUNSKI*, D. GIVEON AND U. Z. LITTAUER Department o[ Biochemistry, Weizmann Institute o[ Science, Rehovot (Israel) (Received October 8th, I969)
SUMMARY M e t h i o n i n e - s t a r v e d t R N A was extracted from a "relaxed" methionine-requiring m u t a n t of Escherichia coli grown in a m e d i u m deficient in methionine. The m e t h y l deficient t R N A T M a n d n o r m a l m e t h y l a t e d t R N A phe species, found in u n f r a c t i o n a t e d m e t h i o n i n e - s t a r v e d t R N A , were separated a n d extensively purified employing chrom a t o g r a p h y on reversed-phase columns, b e n z o y l a t e d DEAE-cellulose a n d m e t h y l a t e d a l b u m i n silicic acid columns. The purified t R N A ph~ species were characterized b y their a m i n o acid accepting capacity a n d e x t e n t of m e t h y l a t i o n . Exposure of n o r m a l E. coli t R N A Ph~ to p H 2. 9 led to changes in its chromatographic p a t t e r n on m e t h y l a t e d a l b u m i n - s i l i c i c acid columns.
INTRODUCTION The s t u d y of the role of m e t h y l a t e d bases in t R N A has been made possible b y the f i n d i n g of MANDEL AND BOREK1,2 t h a t methyl-deficient t R N A is synthesized b y relaxed m e t h i o n i n e - r e q u i r i n g m u t a n t s of Escherichia coli d u r i n g m e t h i o n i n e starvation. t R N A isolated from such a m e t h i o n i n e - s t a r v e d E. coli culture is a m i x t u r e of methyl-deficient t R N A a n d m e t h y l a t e d n o r m a l t R N A . I n the case of m e t h i o n i n e starved t R N A Phe the methyl-deficient a n d n o r m a l species can be resolved b y methylated a l b u m i n - s i h c i c acid c o l u m n c h r o m a t o g r a p h y 3,4 c o u n t e r c u r r e n t d i s t r i b u t i o n s a n d reversed-phase c h r o m a t o g r a p h y s. I n order to s t u d y more closely the properties of methyl-deficient t R N A ~he species a n d compare t h e m to n o r m a l t R N A Phe we have developed procedures for their separation a n d extensive purification. Nomenclature: In this paper methioniue-starved tRNA refers to the tRNA extracted from a relaxed methionine-requiring mutant of E. coli (K12W6) grown in a medium deficient in methionine. Methyl-deficient tRNA refers only to those species which contain few or no methyl groups. Methionine-starved tRNA is a mixture of methyl-deficient tRNA and methylated normal tRNA. tRNAPhe (NN), methylated normal tRNAPhe synthesized under normal growth condition; tRNAPhe (DS), methyl-deficient tRNAPhe isolated from methionine-starved cells; tRNAPhe (NS), normal tRNAPhe isolated from methionine-starved cells; tRNAPhe (DS+NS) methionine-starved tRNAPhe containing a mixture of tRNAPhe (DS) and tRNAPhe (NS). * Present address: Department of Virology, Hadassah Medical School, The Hebrew University, Jerusalem, Israel. Biochim. Biophys. Acta, 199 (197o) 382-393
METHYL-DEFICIENTtRNA Phe
383
MATERIALS AND METHODS
Growth o/ E. coli cells and preparation o/tRNA E. coli K12W6 RC tel m e t - was obtained from Dr. Esther Lederberg. Normal cultures were grown in minimal medium 7 containing o.oi M potassium phosphate buffer (pH 7.4) supplemented with 50/~g/ml of L-methionine at 37 ° with forced aeration. Cells were harvested in the middle of the logarithmic growth phase and the tRNA isolated immediately according to a previously described procedure s, except that the final dialysis step was omitted. The t R N A was then incubated at 37 ° in Tris-HC1 buffer (pH 8.8, 0.2 M) for 3 h. Methionine-starved cultures were grown as follows: a starter culture supplemented with 8/~g/ml of L-methionine was grown overnight with slow shaking. 600 ml of this culture were inoculated into 12 1 of medium supplemented with 4/~g/ml of L-methionine. The culture was grown at 37 ° with forced aeration. After growth had stopped, the culture was further incubated with continued aeration for an additional 3.5 h. All the tRNA preparations were routinely assayed for the presence of nuclease contamination s. Aliquots of the t R N A solution were diluted with I vol. of autoclaved 0.2 M Tris-HC1 buffer (pH 8.8) and held at 37 ° over chloroform for 25 h. The t R N A was then isolated by ethanol precipitation and assayed for its capacity to accept phenylalanine, tRNA preparations showing more than 15 % decrease in their acceptor activity were discarded.
Phenylalanyl-tRNA synthetase Normal E. coli K12W6 cells (2oo g) were suspended in 6oo ml of o.oi M TrisHC1 buffer (pH 7.8) containing o.oi M MgC12 and I/~g/ml of deoxyribonuclease at 4 °. The suspension was homogenized in a Mantan-Gaulin homogenizer (Everett, Mass.) by recycling it 3 times at 4000 lb-inch -2. The lysate was centrifuged for IO rain at 15 ooo ×g and the precipitate discarded. The supernatant fluid was centrifuged at lO5 ooo ×g for 2 h. The lO5 ooo × g supernatant was stirred and o.I vol. of io % streptomycin sulfate solution (freshly prepared and neutralized to pH 7.0) was added. After 30 min of stirring, precipitated material was removed by a Io-min centrifugation at 15 ooo ×g, and the supernatant fraction adjusted to pH 8.0 with 0.2 M NaOH. (NHa)~S Q fractionation was next carried out, and the 30-39 % saturation cut recovered by centrifugation. This protein fraction was dissolved in 60 ml of 0.02 M Tris-HC1 buffer (pH 7.4). To this solution were added: 0.2 vol. of o.I M ATP, 0.2 vol. of o.I M L-phenylalanine, 0.04 vol. of 0.5 M MgC12, o.14 vol. of 0.05 M K F and 0.06 vol. of 0.5 M of K2HPO 4. This mixture was incubated at 55 ° for 45 min, and denatured protein was removed by chilling and centrifugation. 1.6 vol. of saturated (NH4)2SO 4 solution was now added to the supernatant fraction. The precipitate was collected by centrifugation, dissolved in 12 ml of 0.02 M Tris-HC1 buffer (pH 7-4) and dialyzed overnight against the same buffer. Traces of residual nuclease were removed by chromatography through a 2 cm × 20 cm DEAE-cellulose column 9. The column was equilibrated with initial buffer consisting of 0.02 M potassium phosphate l~uffer (pH 7-5), 0.02 M 2-mercaptoethanol and o.ooi M MgC12. The extract was applied, and the column was washed with 500 ml of the initial buffer. The enzyme was eluted with a buffer containing 0.25 M potassium phosphate (pH 6.5), 0.02 M 2-mercaptoethanol, and o.ooi M MgC12. Peak activity fractions were pooled, dialyzed against Biochim. Biophys. Acta, 199 (197o) 382-393
384
~. BIEZUNSKI et al.
o.oi M Tris-HC1 buffer (pH 7.4) and dithiothreitol was then added to a final concentration of 0.25 mg/ml. The enzyme solution was distributed into small aliquots and stored frozen at --15 °. Dialysis bags were boiled twice in 5 % Na2CO3, rinsed with water, then boiled in 0.05 M E D T A (pH 8.0) and stored in o.I mM EDTA.
Amino acid acceptance assay For assay of Phe-tRNA formation, the reaction mixture (o.o5 ml) contained: 2. 7 #moles of Tris-HC1 buffer (pH 7.8); o.I/,mole of ATP; o.I/*mole of GSH (neutralized); I.O/*mole of MgC12; 15o/*/*moles of El*C~phenylalanine (4" lO5 counts/min per m#mole)' 200 ##moles of each of the 19 other 14C-labeled amino acids; O.l-O.2 A26o ms units of unfractionated t R N A or about o.oi A280 m # units of purified t R N A Phe and 12/~g of enzyme protein. After a Io-min incubation at 37 °, the assay tubes were mixed with 0.2 mg of serum albumin, and 3 ml of 5 % trichloroacetic acid was added. After IO rain at 4 ° the precipitates were collected onto membrane filters, washed twice with 5-ml portions of 5 ~o trichloroacetic acid, dried, placed in IO ml of toluene scintillation fluid and radioactivity was counted in a Packard Tri-Carb scintillation spectrometer. For assay of amino acid acceptance of tRNA, a mixed aminoacyl-tRNA synthetase was prepared from E. coli A-I 9 cells according to the procedure of MOENCH AND BERGs up to and including the DEAE-cellulose step. The reaction mixture (0.05 ml) contained: Tris-HC1 buffer, ATP, GSH and MgC12 as detailed above for phenylalanine acceptance assay, and 0.2/~C of a mixture of 15 14C-labeled L-amino acids mixed together with IO re#moles of unlabeled L-phenylalanine; 0.03-0.2 A260ms units of t R N A and 53 #g of enzyme protein. The mixture was incubated for io min at 37 °. The subsequent steps were the same as for the assay of Phe-tRNA. The 14Clabeled amino acid mixture was purchased from New England Nuclear Corp. and contained a mixture of 15 L-amino acids at approx. 40 mC/matom of carbon. I mC of this mixture contained: alanine (80 #C), arginine (7° #C), aspartic acid (80/~C), glutamic acid (125 #C), glycine (4°/~C), histidine (15/~C), isoleucine (5 ° #C), leucine (14o/~C), lysine (60/~C), phenylalanine (80 ~C), proline (50/~C), serine (4° #C), threonine (50/~C), tyrosine (40/zC) and valine (80/~C). The concentration of t R N A was estimated by measuring the absorption at 260 m/~. I m g of t R N A was found to elicit an A260 ms of 30 units in o.oi M NaOH (ref. IO) or, alternatively an A260 ms of 25 units in 0.5 M NaC1. The values measured in N a O H were found to be more accurate, and less variable than those obtained in NaC1, consequently the NaOH values were routinely employed for calculation of the amino acid acceptor capacity of tRNA. Whenever the results are expressed in A260 ms units they were measured in 0.5 M NaC1. A ssay method/or tRNA-methylase E. coli K12W6 cells were grown in a minimal medium 7 containing o.oi M potassium phosphate buffer (pH 7.4) and 0.2 % casamino acids at 37 °. The cells were harvested in the early exponential growth phase, washed once with 0.025 M TrisHC1 buffer (pH 8.0) and frozen until use. A mixed tRNA-methylase was made b y grinding frozen cells with 2.5 times wet weight of alumina and extracting the paste with 2. 4 times wet weight of buffer (o.oi M MgC12; o.oi M Tris-HC1 buffer (pH 7.8); Biochim. Biophys. Acta, 199 (i97o) 382-393
METHYL-DEFICIENTt R N A Phe
385
o.oo2 M GSH (neutralized); I/~g/ml deoxyribonuclease; and IO °/o glycerol) at 4 °. The suspension was centrifuged for 15 min at 15 ooo x g and the supernatant was further centrifuged for 2 h at 15o ooo xg. The supernatant was stirred and 0.087 vol. of neutralized IO % streptomycin sulfate solution was added. After 20 min of stirring, precipitated material was removed b y a Io-min centrifugation at 20 ooo xg. The supernatant (9 ml) was applied onto a Sephadex G-5o column (3 cm x 3 o cm) equilibrated with o.oi M Tris-HC1 buffer (pH 7.4) containing 0.002 M GSH (neutralized) and io % glycerol. The enzyme was eluted with the same buffer, peak activity fractions were pooled, GSH and glycerol were added to a final concentration of o.o15 M and 30 % respectively, and stored at - - 1 5 °. For assay of t R N A methylation the reaction mixture (o.15 ml) contained: 3 /,moles of Tris-HC1 buffer (pH 9.0; the final p H is 8.8); 0.57 #mole of MgC1d o.17 /*mole of GSH (neutralized); 13. 5 m#moles of E14C]methyl-S-adenosyl-L-methionine (6.6. IO4 counts/rain per m/*mole); 20/,moles of ammonium acetate; 1.5- 5/*g of t R N A and 250/*g of enzyme protein. After a 6o-min incubation period at 37 °, the assay tubes were mixed with 0.2 mg of serum albumin, and 3 ml of cold 5 % trichloroacetic acid was added. After io min at 4 ° the precipitates were collected onto membrane filters, washed twice with 5-ml portions of 5 % trichloroacetic acid and 5 times with 5 % trichloroacetic acid solution containing o.oi M sodium pyrophosphate. The membrane were then dried and counted. C o l u m n chromatography
Reversed-phase column chromatography was carried out as described b y KELMERS et al. 11 and KELMERSTM. Two parts of Chromosorb W were suspended in one part of 4 % dimethyldilaurylammonium chloride in isoamyl acetate and poured into a jacketed glass column (2.5 cm x 19o cm containing 0.45 M NaC1, o.oi M MgC12 and o.oi M sodium acetate buffer (pH 4-5) saturated with isoamyl acetate at 37 ° (initial buffer). The t R N A was dissolved in IO ml of initial buffer and applied to the column. Linear gradient elution was carried out at 37 ° using 1.8 1 of initial buffer and 1.8 1 of a solution containing 0.75 M NaC1, o.oi M MgC12 and o.oi M sodium acetate buffer (pH 4-5) saturated with isoamyl acetate. The column eluate was collected at a flow rate of 60 ml/h, and o.o4-ml aliquots were removed and assayed for amino acid acceptance. Benzoylated diethylaminoethyl-cellulose-column chromatography was carried out as described by GILLAM et al. 13. The benzoylated diethylaminoethyl-cellulose column (0. 9 cm x 95 cm) was washed with a solution consisting of 2 M NaC1 and 25 % ethanol and then equilibrated with a solution 14 containing 0.4 M NaC1 and 0.05 M sodium acetate buffer (pH 5.0) (initial buffer). The t R N A solution was applied to the column then washed with 7 ° ml of initial buffer and linear gradient elution was carried out as indicated. Methylated albumin-silicic acid columns (2 c m x 12 cm) were prepared as previously described 4. To enhance the methylated albumin-silicic acid-column resolution the labeled Phe-tRNA samples were mixed together with 4 mg of uncharged normal tRNA. The t R N A solution (i.o ml) was diluted with 9.0 ml of 0.8 M NaC1 solution containing 0.05 M sodium acetate buffer (pH 5.5) (initial buffer) and applied to the column. The column was washed with 50 ml of initial buffer and the t R N A was then eluted at 16 ° with a linear gradient containing 200 ml of initial buffer and 200 ml of Biochim. Biophys. Acta, 199 (197o) 382-393
386
N. BIEZUNSKI et al.
i.o M NaC1 solution containing o.o5 M sodium acetate buffer (pH 5.5). Fractions of 3.o ml each were collected at a flow rate of 3o ml/h. Each receiving tube contained o.2 ml of I M sodium acetate buffer (pH 4.5). Assay of acid insoluble radioactivity was carried out as previously described 4. Throughout the purification of t R N A care was taken not to introduce nucleases b y using sterile buffer solutions and glassware. Glass columns were washed with o.I M NaOH, o.I M HC1 and water before use. MATERIALS
Dimethyldilaurylammonium chloride (Aliquat 2o4) was a gift from the Chemical Division of General Mills, Ill., and was recrystallized 3 times from acetone at --2o °. Chromosorb W (acid-washed, dimethyldichlorosilane-treated, lOO-2Oo mesh size) was obtained from Johns Manville. Benzoylated diethylaminoethyl-cellulose ( 5 o - I o o m e s h ) and L@4Clphenylalanine (255 mC/mmole) were purchased from Schwartz BioResearch. [14C]Methyl-S-adeno.,yl-L-methionine (36 mC/mmole) was obtained from Tracerlab. Membrane filters (MF-5o) with pore size of 0.6 # were purchased from Membranfiltergesellschaft, G6ttir.gen. Levigated alumina was obtained from Norton Co. and glycerol from Matheson, Coleman and Bell. RESULTS
Puri/ication o/ normal and methyl-deficient t R N A Phe Reversed-phase chromatography of methionine-starved t R N A resolves two peaks of t R N A Phe (Fig. Ib). The first peak corresponds to methyl-deficient tRNA Phe
0.4 ~
20.0
tRNA phi (NN) I0.
~000
//
ZL
~
o
=
o
tO00
1.0 E
o
I
ea
J
,
'
" -
l
o.e =-
....
zo.o
7ili
2.0
1.0
NA ~ (DS)
0.4 ~
,3000
!10000
~ " =
(NS)
-
z;O0
BOO
3
:oooo
1200
Effluent
1600
2000
.5000
2400
volume ( m l )
F i g . i. R e v e r s e d - p h a s e c o l u m n c h r o m a t o g r a p h y of n o r m a l a n d m e t h i o n i n e - s t a r v e d t R N A . a. I7O m g of n o r m a l t R N A w e r e a p p l i e d t o t h e c o l u m n a n d 9 - m l f r a c t i o n s w e r e c o l l e c t e d , b. 1 8 o m g o f methionine-starved tRNA were applied to the column and 8-ml fractions were collected. Q-O, phenylalanine acceptance (o.o4-ml aliquots); - - , A 260 my.
Biochim. Biophys. Acta, 199 (197 o) 3 8 2 3 9 3
MLTHYL-DEFICIENT t R N A Ph~
387
(DS), while the second peak represents normal t R N A vhe (NS) (see below). This separation is similar to that obtained b y SHUGARTet al. e. I t was also consistently observed that the methyl-deficient t R N A Phe (DS) had a tendency to separate into two components, these were resolved on methylated albumin-silicic acid-column chromatog:aphy (see below). The elution profile of methylated normal tRNA, isolated from log-phase cells grown in the presence of an excess of methionine, is shown in Fig. Ia. The major peak corresponds to normal t R N A vh~ (NN), while a trace of methyl-deficient t R N A Phe is also evident. The peak fractions containing t R N A T M were pooled, concentrated and freed of salts by ultrafiltration. The concentrated t R N A Phe fractions were then subjected .
L6
(0)]
l 0e ~,
112°°
0O8
1
400
0.04
1"6i 0.8
~ - - "~ - --
0,8 u:
16000
0.6 ~
12000
o.4
8o00
0.2
4000 u
16
~
(c:
~0~ o.oe
0.04 I'L
500 t 20
40
60
80 Tube
I00
120 140 160
number
Fig. 2. Benzoylated diethylaminoethyl-cellulose column c h r o m a t o g r a p h y of enriched tRI~APhe fractions, a. tRNAPhe (NN) fractions (between 1575 to 1638 ml, Fig. Ia) were pooled and concentrated b y ultrafiltration t h r o u g h a U M - I o m e m b r a n e (Diaflo ultrafiltration cell Amicon Corp., Lexington, Mass.) to a volume of 4 ml. The c o n c e n t r a t e d solution was diluted with 4 ° ml of w a t e r and t h e n c o n c e n t r a t e d again to a b o u t 2.0 ml. The t R N A solution was applied to the column and w a s h e d w i t h 7 ° ml of o. 4 1V[ NaCI solution containing 0.o 5 M sodium acetate buffer {pH 5.0) (initial buffer). The linear gradient elution was carried o u t at 4 ° using 2oo ml of initial buffer and 200 ml of I . t M NaC1 solution containing o.o 5 M s o d i u m acetate buffer (pH 5.0). This was followed b y a second gradient containing 5 ° ml of i . I M NaC1 and 5 ° ml of 1. 5 M NaC1 containing o.o 5 M s o d i u m acetate buffer (pH 5.0). 4-ml fractions were collected at a flow rate of 36 ml/h. F r a c t i o n s No. 9 4 - t o 2 were pooled, concentrated b y ultrafiltration to a b o u t 4 ml, t h e n diluted w i t h 40o ml of w a t e r and c o n c e n t r a t e d again to a b o u t 2.0 ml. b. The pooled and concentrated tRNAPhe (DS) fractions (between 11o 4 to 12o8 ml, Fig. lb) were applied to the c o l u m n and eluted as described u n d e r a. 4-ml fractions were collected at a flow rate of 48 ml/h. Fractions No. 84-95 were pooled, concentrated and freed of salt as described above, c. The pooled and concentrated tRNAPhe (NS) fractions (between 1688 to 1792 ml, Fig. Ib) were applied to the c o l u m n and eluted as described u n d e r a except t h a t the second eluting gradient consisted of ioo ml of I . I M NaC1 and IOO ml of 1.5 M NaC1 containing 0.o5 M s o d i u m acetate buffer (pH 5.o). 4-ml fractions were collected at a flow r a t e of 48 ml/h. Fractions No. 94-115 were pooled, c o n c e n t r a t e d and freed of salt as described above. Q - Q , phenylalanine acceptance (o.o4-ml aliquots); , A260 me*.
Biochim. Biophys. Acta, 199 (t97 o) 382-393
N. BIEZUNSKI et al.
388
to benzoylated diethylaminoethyl-cellulose chromatography (Fig. 2). In the case of t R N A vhe (NN), and possibly t R N A vhe (NS), the remain tRNA TM peak was found to be followed b y a smaller shoulder. Table I shows the extent of amino acid acceptance of the various t R N A vhe fractions. The combination of reversed-phase column chromatography and benzoylated diethylaminoethyl-cellulose-column chromatography yielded highly purified t R N A vh~ preparations exhibiting a phenylalanine acceptance value of 30-36 m/~moles per mg of t R N A (or 12oo-144o #/zmoles per A2s0 ma unit). Further attesting to the high degree of purification we have achieved is the decidedly reduced ability of all preparations to accept amino acids.
TABLE I PURIFICATION
OF N O R M A L A N D
METHIONINE-STARVED tl~NA phe
Sample
Initial normal tRNAPhe (NN) Reversed phase of tRNAPhe (NN) Benzoylated diethylaminoethylcellulose of tRNAPhe (NN) Initial methionine-starved tRNAPhe (DS+NS) Reversed phase of tRNAPhe (DS) Benzoylated diethylaminoethylcellulose of tRNAPhe (DS) Reversed phase of tRNAPhe (NS) Benzoylated diethylaminoethylcellulose of tRNAPhe (NS)
Methyl groups incorporated (mttmoles/mg RNA )
A mino acids incorporated Phenylalanine (mlzmoles/mg RNA )
Amino acid mixture* (counts/rain per i~g R N A )
o.41 1.44
1.25 14. 5
388o 191o
0.77
30.2
720
45.9 49.7
1.47 21.6
442o 154o
6o. 4 18.9
35.9 2o. 4
48o 148o
lO. 7
29.7
465
* Assayed with crude aminoacyl-tRNA synthetase and a mixture of 15 14C-labeled L-amino acids mixed together with unlabeled L-phenylalanine.
The methyl group accepting capacities of the t R N A Phe fractions were assayed with a mixture of crude tRNA-methylase from E. coli (Table I). When normal t R N A Ph~ (NN) was used as methyl acceptor very little methylation occurred; on the other hand with methyl-deficient t R N A Phe (DS) a substantial amount of methyl groups were incorporated. The normal t R N A Phe (NS) fraction isolated from the unfractionated methionine-starved t R N A showed a significant ability to accept methyl groups and seems still to be contaminated with some species of methyl-deficient t R N A other than t R N A TM.
Methylated albumin-silicic acid-column chromatography of the purified t R N A Phe fractions The identity of the various t R N A Phe fractions was verified b y methylated albumin-silicic acid-column chromatography 4. Fig. 3 a shows the elution profile of unfractionated [14ClPhe-tRNA (NN) and unfractionated methionine-starved [sH]Phe Biochim. Biophys. Acta, 199 (197 o) 382 393
389
METHYL-DEFICIENT t l ~ N A Phe I
I
I
I
I
1
{o)
U nfroclionoted
[~'H]phl"IRNA{0S*NS) L6
--
/
1,2 0.8
I
I
I
(b)
UnfractionQted [3H]P.e-tRNA (NN)
3H
14C
I
1800
6 0 0 0 I'-
~(NN)
3H
~/ Pu,ili,d
4000 t4000
12ooo !2000
,oooooVtI
Oa
Unfroclionoted
(c)
~.3H]Phe-tRNA(05÷N$) Purified
12
E
"~-
I I I I UnfractioneO
]
I (d)
[3HIPhe_l RNAtOS+NS ) 3H I'4Cfi-~ ,~! :! [:Ooi]l~i ,~i'~dN A(NS) 3000
, :i.
14C
6000
.
,~ '.
:
3H
14c
~.000
/
6000
i! 2000
0.8
,ooo
0.4
20
40
60
80
ooo
I00
2000
20 Tube
40
60
80
I00
number
Fig. 3. Methylated albumin silicic acid-column chromatography of P h e - t R N A fractions, a. O - O , 12o ooo counts/rain of unfractionated normal [14C]Phe-tRNA (NN); O - - -O, ioo ooo counts/min of unfractionated methionine-starved ~ H ] P h e - t R N A (DS + N S ) . b. O - Q , 7 ° ooo counts/rain of purified [14C]Phe-tRNA (NN) fractions (No. 94-1o2, Fig. 2a); O - - - O , IOO ooo counts/rain of unfractionated normal F3H]Phe-tRNA (NN). c. 0 - 0 , 220 ooo counts/min of purified []4C]Phet R N A (DS) fractions (No. 84-95, Fig. 2b); O - - - O , I7OOOOCounts/min of unfractionated methionine-starved [3H]Phe-tR~A ( D S + N S ) . d. Q - O , ioo ooo counts/min of purified [14CJPhet R N A (NS) fractious (No. 9 4 - i I ~ , Fig. 2c); O - - - O , 225 ooocounts/min of unfractionated methionine-starved [~H]Phe-tRNA ( D S + N S ) . All labeled l~he-tRNA samples were mixed together with 4 mg of uncharged normal t R N A (unlabeled); - - , A 26~ mr*.
tRNA (DS+NS) on a methylated albumin-silicic acid column. The methioninestarved [~H]Phe-tRNA resolved into three main peaks. The first two peaks eluting from the methylated albumin-silicic acid column correspond to methyl-deficient Phe-tRNA (Peaks a and b in Fig. 2, ref. 4) while the last peak corresponds to normal Phe-tRNA (Peak d mixed together with Peak c in Fig. 2, ref, 4). In some experiments an additional peak was observed (Peak c in ref. 4) which preceded the major peak of normal Phe-tRNA. However, its resolution was variable and depended on the particular batch of methionine-starved tRNA as well as on the sample of methylated albumin-silicic acid used for the column chromatography. The assignments of the methylated albumin-silicic acid-column peaks were verified by rechromatography on methylated albumin-kieselguhr columns, on which the nature of the peaks had already been established in an earlier communication 1~. Fig. 3b shows that purified [14C]Phe-tRNA (NN) and unfractionated [3H]Phe-tRNA (NN) coeluted from the methylated albumin-silicic acid column. Purified methyl-deficient EI*C]Phe-tRNA (DS) fraction resolved into two species (Fig. 3c) that appear to coincide with the first two peaks of methionine-starved [~H]Phe-tRNA (DS+NS). The behavior of purified [14C]Phe-tRNA (NS) upon methylated albuminsilicic acid-column chromatography is depicted in Fig. 3. The results indicate that Biochim. Biophys. Acta, 199 (197 o) 382-393
390
N. BIEZUNSKI et al.
this fraction has similar chromatographic properties to the last peak of unfractionated methionine-starved Phe-tRNA as well as to Phe-tRNA isolated from E . coli cells grown under normal conditions. Since reversed-phase chromatography necessitated prolonged exposure of tRNA at pH 4.5 it was important to examine whether this treatment had any effect on this tRNA. Unfractionated methionine-starved tRNA was therefore incubated in the presence of the terminal buffer (0.75 M NaC1, o.oi M MgC12 and o.oi M sodium acetate buffer (pH 4.5) saturated with isoamyl acetate) for a period of 4 days at 37 °. The tRNA was then isolated by ethanol precipitation and its ability to accept phenylalanine was assayed. Only a slight decrease of activity was observed (1.24 vs. 1.51 m/zmoles of phenylalanine per mg RNA for the incubated and unincubated preparations respectively). Methylated albumin-silicic acid-column chromatography of the incubated E14C~Phe-tRNA together with the unincubated I~HIPhe-tRNA showed that both preparations had the same elution profile. Thus, by the methylated albumin-silicic acid-column criteria no adverse effects have occurred during the incubation of the tRNA at pH 4.5. It was, however, noted that exposure of tRNA to a lower pH did affect both its phenylalanine acceptor ability and its chromatographic properties on methylated albumin-silicic acid columns. Incubation of unfractionated normal tRNA at pH 2. 9 for 2 h at 37 ° according to the procedure of THIEBE AND ZACHAU16 resulted in a significant loss of activity (0.50 vs. 0.86 m#moles phenylalanine per mg RNA for the incubated and unincubated preparations respectively). In addition, methylated albumin-silicic acid-column chromatography revealed an altered elution profile for the pH-2. 9 treated Phe-tRNA. I
I
I
I
I
i /['4c]Phe-t~NA
"C
pH 2.9 treoted
000
i/i
~_.
~4000
I
1
.°° Phe-tRNA
0
12
I
600
0.8
400
Z400
E
-~16oo ~ /
20
40
60
80 Tube
I00
120
140
160
number
Fig. 4" Methylated albumin-silicic acid-column c h r o m a t o g r a p h y of pH-2.9 treated n o r m a l P h e - t R N A . 2 ml of u n f r a c t i o n a t e d n o r m a l t R N A (5 mg) was dialyzed overnight against w a t e r at 4% a d j u s t e d to p H 2.9 with a m m o n i u m f o r m a t e buffer (final concn, o.i M) and incubated at 37 ° for 2 h. The t R N A solution was a d j u s t e d to p H 7.4 with I M Tris-HCl buffer (pH 8.8) and an aliquot was r e m o v e d and charged with Ex4C]phenylalanine. The pH-2.9 t r e a t e d CxIC]Phe-tRNA (27 5oo c o u u t s / m i n , ( D - G ) was mixed t o g e t h e r with u n t r e a t e d normal [ 3 H ] P h e - t R N A (60 ooo c o u n t s / m i n , O - O ) a n d 8 m g of carrier uncharged n o r m a l t R N A and applied to a m e t h y l a t e d albumin-silicic acid column. - - -, A 2n3 mu.
Biochim. Biophys. Acta, 199 (I97 o) 382-393
METHYL-DEFICIENT tRNAph~
391
Fig. 4 shows that following p H 2. 9 treatment two additional peaks of Phet R N A appear that are more retarded on the methylated albumin-silicic acid column than the main, normal Phe-tRNA component.
DISCUSSION
The ability of methyl-deficient t R N A to accept amino acids was studied in several laboratories. Early experiments 17-23 demonstrated that unfractionated methionine-starved t R N A preparations (consisting of a mixture of methyl-deficient and normal methylated tRNA) incorporated several amino acids to the same extent as fully methylated normal tRNA. In contrast to the above studies SHUGART et al. ~a reported that the extent of incorporation of four amino acids, namely phenylalanine, leucine, tyrosine and histidine, was decreased in unfractionated methionine-starved t R N A preparations. In addition, this group assayed the phenylalanine-accepting capacity of normal and methyl-deficient t R N A vhe separated by reversed-phase column chromatography. They found s that the total amount of phenylalanine accepted as well as the initial velocities oi phenylalanine incorporation correlate with the degree of undermethylation. In the present studies we have compared the phenylalanine accepting capacity of unfractionated normal and methionine-starved tRNA. Though the specific activities varied from one preparation to another, most of our unfractionated methioninestarved preparations showed a somewhat higher activity (lO-25 %) than the normal preparation. In none of the m a n y cases examined had the methionine-starved t R N A a lower activity than the normal tRNA. Table I shows that the methyl-deficient t R N A vhe species can be purified extensively. The purification that we have achieved (35.9 m#moles per nag RNA) would indicate that the methyl-deficient t R N A vhe is almost pure. Thus, under the condition of our assay system there is no indication t h a t the extent of phenylalanine acceptance of the methyl-deficient t R N A vhe is lower than that of the normal t R N A vh" species. It should be stressed that the success of the purification procedure was found to depend on the availability of t R N A preparations that are free of nuclease contamination (see METHODS), it also depended on the use of sterile buffer solutions and glassware. It was shown in the present studies that prolonged exposure of t R N A vhe in the pH-4.5 buffer, used for the reversed-phase chromatography, did not affect its phenylalanine accepting capacity or its chromatographic behavior on methylated albumin-silicic acid columns. As shown in Fig. 3, the purified Phe-tRNA (NN), Phe-tRNA (DS) and Phe-tRNA (NS) coeluted with the corresponding components of the unfractionated tRNA. Thus, b y the methylated albumin-silicic acid-column criteria no adverse effects have occurred during the purification of the t R N A vh" fractions. By contrast, is was found that exposure of t R N A vhe to a lower p H value, namely p H 2.9, does reduce its activity as well as alters its elution properties from methylated albumin-silicic acid columns. The pH-2.9 treatment was found by THIEBE AND ZACHAUle to excise from yeast t R N A phe the base Y+ which is located right next to the anticodon 25. The effect of the pH-2.9 treatment on E . coli t R N A vh*, however, is not due to excision of the base Y+ since the latter is absent from this t R N A species l°,zn. The methylated albumin-silicic acid elution profile of pH-2.9-treated Biochim. Biophys. Acta, I99 (I97o) 382-393
392
N. BIEZUNSKI et al.
tRNA Phe also does not correlate with the change in the methylated albumin-kieselguhr-column chromatographic profile of aged, normal Phe-tRNA preparations 15,3. In the latter case storage of frozen Phe-tRNA preparations resulted in the appearance of a new peak eluting from the methylated albumin-kieselguhr column at a lower salt concentration than the major Phe-tRNA peak. Whether the pH 2. 9 treatment affects the secondary structure of tRNA Phe or one of its minor bases remains to be determined. The inclusion of glycerol throughout the isolation procedure of the tRNAmethylase, was found to considerably enhance its activity. Thus, the extent of methylation of methionine-starved tRNA was increased by 2-2.5-fold. It was observed that about 50 % of the expected methyl acceptor sites of purified methyl-deficient tRNA Phe (DS) were methylated (60 vs. 113 m/~moles of methyl groups per mg RNA). These results may indicate that the purified methyl-deficient tRNA Ph~ species still contains a significant amount of methyl groups or that our crude tRNA-methylase was unable to methylate all the available acceptor sites. It should also be recalled that the methyl-deficient tRNA Pae (DS) can be resolved by methylated albuminsilicic acid-column chromatography into two components (Fig. 3). These may differ in their primary sequence or their degree of methylation. Another possibility is that these two methyl-deficient tRNA Ph~ components differ in the degree of oxidation of their thiolated bases or tile extent of covalent bond formation a7 between the 4-thiouridine in Position 8 and the cytidine residue in Position 13 resulting from the influence of ultraviolet light on methyl-deficient tRNA Ph*.
ACKNOWLEDGMENT
This research was supported, in part, by agreement Institutes of Health, U.S. Public Health Service.
No. 455114
of the National
REFERENCES I L. R. MANDEL AND E. BOREK, Biochem. Biophys. Res. Commun., 4 (1961) 14. 2 L. R. MANDEL AND E. BOREK, Biochemistry, 2 (1963) 560. 3 U. Z. LITTAUER, M. REVEL AND R. STERN, Cold Spring Harbor Syrup. Quant. Biol., 31 (1966) 5Ol. 4 R. STERN AND U. Z. LITTAtlER, Biochemistry, 7 (1968) 3469 . 5 E. FLEISSNER, Biochemistry, 6 (1967) 621. 6 L. SHUGART, G. D. ~NTOVELLIAND M. P. STULBERG, Biochim. Biophys. Acta, 157 (1968) 83. 7 A. D. HERSHEY AND M. CHASE, J. Gen. Physiol., 36 (1952) 39. 8 U. Z. LITTAUER, S. A. YANKOFSKY, A. NOVOGRODSKY, H. BURSZTY'N, Y. GALENTER AND E. KATCHALSKI, Biochim. Biophys. Acta, 195 (1969) 29. 9 K. H. MUENCH AND 12). BERG, in G. L. CANTONI AND D. R. DAVIES, Procedures in Nucleic Acid Research, Harper and Row, New York, 1966, p. 375. IO P. BERG, F. H. BERGMANN,E. J. OFENGAND AND 1~I. DIECKMANN, J. Biol. Chem., 236 (1961) 1726. I I A. D. KELMERS, G. D. NOVELLI AND M. P. STULBERG, J. Biol. Chem., 240 (t965) 3979. 12 A. D. KELMERS, [. Biol. Chem., 241 (1966) 354 o. 13 I. GILLAM, S. 1V[ILLWARD, D. BLEW, M. VON TIGERSTROM, E. WIMMER AND G. M. TENER, Biochemistry, 6 (1967) 3043 . 14 K. L. RoY AND D. S6LL, Biochim. Biophys. Acta, 161 (1968) 572. 15 M. }{EVEL AND U. Z. LITTAUER, Biochem. Biophys. Res. Commun., 20 (1965) 187. 16 JR. THIEBE AND H. G. ZACHAU,European J. Biochem., 5 (1968) 546. 17 F. C. NEIDHARDT AND L. EIDLIC, Biochim. Biophys. Acta, 68 (1963) 380. 18 J. L. STARR, Biochem. Biophys. Res. Commun., io (1963) 181.
Bio=him. Biophys. Acta, 199 (197 o) 382-393
METHYL-DEFICIENT t R N A Ph"
393
19 I. SVENSSON, H. G. BOMAN, K. G. ERIKSSON AND K. KJELLIN, J. Mol. Biol., 7 (1963) 254. 20 U. Z. LITTAUER, K. H. MUENCH, P. BERG, W. GILBERT AND P. F. SPAHR, Cold Spring Harbor Syrup. Quant. Biol., 28 (1963) 157. 21 A. PETERKOFSKY, C. JESENSKY, A. BANK AND A. H. MEHLER, J. Biol. Chem., 239 (1964) 2918. 22 U. Z. LITTAUER, Proc. 6th Intern. Congr. Biochem., 32 (1964) i i . 23 U. Z. LITTAUER AND R. MILBAUER, in Syrup. on R N A Structure and Function, Proc. 2nd Meeting Federation European Biochem. Socs., Vol. 4, P e r g a m o n , London, 1965, p. 9. 24 L. SHUGART, B. H. CHASTAIN, G. D. NOVELLI AND M. P. STULBERG, Biochem. Biophys. Res. Commun., 31 (1968) 404 . 25 W. L. RAJBHANDARY, S. H. CHANG, A. STUART, R. D. FAULKNER, t~. IV[. HOSKINSON AND H. G. KHORANA, Proc. Natl. Acad. Sci. U.S., 57 (1967) 75126 B. G. BARRELL AND F. GANGER, F E B S Letters, 3 (1969) 275. 27 M. YANIV, A. FAVRE AND B. G. BARRELL, Nature, in the press.
Bioehim. Biophys. Acta, 199 (197 o) 382-393