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Restoration of methionine-accepting and transformylation activity by combining oligonucleotide fragments derived from a ribonuclease T1 digest of Escherichia coli tRNAfMet
408
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY NOTES BBA 91238
Restorotion of methionine-accepting ond tronsformylation octivity by combining oligonucleotide fragments derived from o ribonucleose TI digest of Escherichia coli t R N A fM°t Since the validity of the "cloverleaf" model 1 has been supported by investigations of a number of t R N A species whose nucleotide sequences have been established and since all of the physical and chemical evidence available is compatible with the cloverleaf arrangement 2, it would now be reasonable to discuss the nature of the t R N A molecule on the basis of this model. Using the 5'- and 3'-halves obtained by splitting the I - A bond of the presumed anti-codon, IAC of yeast t R N A val, BAYEV et al. 3 showed that the two halves restored the total valine-acceptor activity of the initial molecule only when mixed. The 5'and 3'-halves were obtained in our laboratory 4 from Escherichia coli t R N A wl, and they retained the valine-acceptor activity only when they were mixed and renatured. Preliminary data suggest that the cleavage occurred at the presumed anti-codon (unpublished results). This is good evidence that the aminoacyl-tRNA synthetase does not necessarily require the intactness of the anti-codon sequence in those t R N A molecules. As for the left-hand dihydrouridine loop, it could be a recognition site for the synthetase, since this loop is sufficiently different in all of the sequences determined so far 2. In this paper we show that t R N A ~Met from E . coli strain B could be split into two at the dihydrouridine loop region when the t R N A molecules were digested by ribonuclease T 1 in the presence of Mg *+ and that the resulting two halves restored the methionine-accepting and the transformylation ability when they were mixed. Thus the intactness of the dihydrouridine loop is not required either for the synthetase or the transformylase. The t R N A fMet was purified b y chromatography on a column of DEAE-Sephadex followed by chromatography on BD-cellulose, as reported previously 5 (crude t R N A was kindly prepared for us by Dr. S. ESUMI of Kaken Chemicals). 2o0 A260 m# units of the t R N A were dissolved in io ml of o.I M Tris-HC1 (pH 7.5) and 0.02 M magnesium acetate, and incubated at 37 ° for 26 rain together with 527 #g of ribonuclease T 1 (a kind gift of Dr. H. OKAZAKI of Central Research Laboratories, Sankyo Co.). The ribonuclease and hydrolysed products were removed from the partially digested t R N A by the method described previously 6. 97 A 26o m# units of the material were recovered. 14.5 % of the initial methionine-acceptor activity remained in this preparation which was heated in the presence of o.oi M EDTA (pH 7.o), 7 M urea and 0.02 M Tris-HC1 (pH 7.2) in a total volume of 3 ml at 80 ° for 4 rain, then rapidly cooled and subjected to a DEAE-Sephadex column chromatography at p H 2.7. As shown in Fig. I, the elution profile gave three peaks and one shoulder, which were named Fractions K, L, M and N, respectively. The materials were collected from each fraction by precipitation with 3.5 vol. of ethanol at --20 ° overnight followed b y centrifugation and dialysed against 0.05 M NaC1 and 0.02 M Tris-HC1 (pH 7.5) and then water to remove urea and salts. The methionine-accepting and the transformylation activities were assayed for each peak and combinations of t R N A fMet fragments Biochim. Biophys. Acta, 174 (1969) 4o8-411
409
PRELIMINARY N O T E S t
J
K L M
t
N
o
~0.08
L
25
o
510 F r a c t i o n No.
7"5 . . . .
- 1010
zL
'~
Fig. I. C h r o m a t o g r a p h i c s e p a r a t i o n of tRNAfMet f r a g m e n t s . 95 Azeo mu u n i t s of the ribonuclease Tl-treated tRNAfMet were applied to a D E A E - S e p h a d e x c o l u m n (0.5 cm × ioo cm) pre-equilib r a t e d w i t h 7 M urea solution (pH 2.7). The linear g r a d i e n t elution w a s carried o u t a t 20 ° using I5o ml of o.6 M NaC1 and 7 M u r e a (pH 2.7) in the reservoir a n d 15o ml of 7 M u r e a (pH 2.7) in the mixing vessel. The elution w a s complete w i t h i n 16 h. E a c h fraction contained 3 ml of the effluent. A b s o r b a n c e at 260 m/, ( 0 ) and at 330 m/* ( O ) was measured. The materials collected f r o m F r a c t i o n s 44-47, 49-52, 54-58 and 59-67 were designated F r a c t i o n s K, L, M and N, repectively.
TABLE I RESTORATION OF M E T H I O N I N E - A C C E P T O R
AND TRANSFORM'YLATION ACTIVITIES BY COMBINING
tRNAIMet FRAGMENTS O.O15 A2e0 mg u n i t of each f r a g m e n t as well as the i n t a c t t R N A was dissolved in a final v o l u m e of o.i ml of o.2 M NaC1, o.oi M MgC1 a and o.o2 M Tris-HC1 (pH 7.2) a n d i n c u b a t e d at 37 ° for i h. Methionine-acceptor 2 a n d the t r a n s f o r m y l a t i o n 8 activities were assayed as described previously.
Fragment
p4C]Methionine * accepted (counts/rain per o.o2 ml)
K L M N
5 8 133 lO 3
K+M K+lq L+M L+Iq M+lq
282 197 468
[14C] Formate** accepted
(counts/min per o.o4 ml) 2 o 37 28
Net increase
i334 516
144 89 327 i223 28o
I n t a e t R N A f M e t 1153
Net increase 56 52 122 360 I12
17 22
85 332 47
382
" Specific activity, 218 m C / m m o l e . ** Specific activity, 33 m C / m m o l e .
Biochim. Biophys. Acta, I74 (1969) 4 o 8 - 4 I I
410
PRELIMINARY NOTES
as presented in Table I. The results show that both activities were recovered to about 50 % of those of the native t R N A f~a only when the L and the N were mixed. Although not shown in Table I, the mixing of the K, L and M fractions did not give rise to the recovery, nor did the addition of the K and/or the M fraction to the mixture of the L and the N fractions inhibit the restoration. I t is interesting to know the nucleotide sequences of the oligonucleotide present in the L and N fractions. Since the complete nucleotide sequence of t R N A fM~t from E. coli has been proposed 9 and the column chromatographic elution pattern of the ribonuclease T,-digested t R N A ~iet is known (ref. 5 and unpublished data), 2 A2~0 m/, units each from Fractions L and N were subiected to chromatography to obtain the oligonucleotide compositions of the two fragments, as shown in Fig. 2. Although the L and N fractions are not very pure, 0
0025
0.015
4
= 0,005
.._/
E o
OFb
0.00
i'
0['-" %
o
100
200
300
400
500
Effluent vol. (ml)
Fig. 2. C h r o m a t o g r a p h y of a complete ribonuclease T x digest of tRNAiMet, the L f r a g m e n t and the N fragment. Samples were r u n on a D E A E - S e p h a d e x A-25 c o l u m n (o.55 cm × 15o cm) at n e u t r a l p H w i t h an NaC1 gradient f r o m o.14 to o. 7 M (4oo ml × 2) in the presence of 7 M urea, as described previously 5. (a) 3 A2e0 m~ u n i t s of t R N A f i e t , (b) 2 A2~0 m~ u n i t s of t h e L and (c) 2 A~6~ ma u n i t s of t h e N were used. The elution profile w a s o b t a i n e d in a cell w i t h a 4 - m m light p a t h b y a u t o m a t i c ultraviolet recorder of Model JLC-3BC ( J a p a n E l e c t r o n Optics L a b o r a t o r y Co.) w i t h a filter (~max, 260 m/g;dJtllZ, 7 m/~). The flow r a t e w a s 0.3 ml/min, mTG = 7-methylguanosine; t*U = 4-thiouridine; Cm = 2'-O-methylcytidine.
the following points could be seen from the comparison of the relative amounts of peaks obtained from the L and N fractions and the intact tRNA. The L and N fractions both possess one of the two tetranucleotides (Peak 6), since the L gave half the amount of Peak 6 given by intact tRNA, and the N fraction gave Peak 6 the amount of which is almost identical to Peak 2 which is comprised of two dinueleotides. These facts indicate that the cleavage occurred at the dihydrouridine region if judged on the basis of the t R N A fMet arranged in the cloverleaf pattern (see ref. 9). The following observations from the chromatograms are all compatible with the above judgement, leading to the conclusion that the L fraction represents the 3'half and the N the 5'-half of the tRNAfMet: (i) Many more Gp are in the N fraction. (2) Dinueleotides are not in the L but in the N fraction. (3) 4-Thioufidine is in the N fraction, as iudged b y its specific absorbance at 33o m#, shown in Fig. I (*U in Biochim. Bgophys. Acta, 174 (1969) 4o8-411
PRELIMINARY NOTES
411
the sequence established by DUBE et al2 was identified as 4-thiouridine: see ref. 5). (4) 7-Methylguanosine-containing tetranucleotide is in the L fraction. (5) Hexanucleotide-containing CCA terminus is in the L but not in the N fraction. (6) (Cp).~Gp and the two large fragments (Peaks 8 and 9) were in the L fraction. Detection of the minor components, including pCp, by the two-dimensional paper chromatography of the ribonuclease T 2 digest s of the L and N fractions also agreed with the above observations. Although not shown in Fig. I, the K fraction turned out to be the anti-codon loop fragment consisting of a nonadecanucleotide as reported by CLARK, DUBE AND MARCKER1° and the M fraction possessed all of the sequence of the L fraction except the hexanucleotide containing the CCA terminus. The maximum methionine-accepting activity was observed when the L and N fractions were mixed in a ratio of 2:1. This ratio agreed closely with the theoretical ratio, 2.3:1 , which was calculated with the aid of the molar extinction coefficient and the assumption that the sphtting occurled at -DpApGpCpUp- (D -- dihydrouridine) (see ref. 9). It should be mentioned that the restoration of those biological activities did not require particular conditions (temperature, salt concentration or pre-incubation period) just as for tRNA val from yeast z, while in the case of E. coli tRNA wl, the recovery depended on pre-incubation temperature, period and salt concentration 4. In conclusion, the L and N fractions are the 3'- and 5'-halves, respectively, of the tRNA fMet which result from splitting somewhere in the dihydromidine loop region. The methionine-accepting activity and the transformylation ability were retained only when the two halves were mixed. Determination of the precise site of the break in the dihydrouridine loop region and further study on the other biological properties of the fragments are now in progress.
Virology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)
TAKESHI SENO MASARU KOBAYASHI SusoMu NISHIMURA
I R. J. 2 J. 3 A. 4 5 6 7 8 9 IO
W. HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, M. MARQUISEE, S. H. MERRILL, R. PENSWICK AND A. ZAMIR, Science, 147 (1965) 1462. T. MADISON, Ann. Rev. Biochem., 37 (1968) 131. A. BAYEV, I. FODOR, A. D. MIRZABEKOV, V. D. AXELROD AND L. Y. KAZARINOVA, Molekularnaya Biologiya, I (1967) 859. K. ODA, F. KIMURA, F. HARADA AND S. NISHIMURA, Biochim. Biophys. Acta, in t h e press. T. SENO, M. KOBAYASHI AND S. NISHIMURA, Biochim. Biophys. Acta, 169 (1968) 80. S. NISHIMURA AND G. D. ~OVELLI, Proc. Natl. Acad. Sci. U.S., 53 (1965) 178. S. lX]'ISHIMURA, F. HARADA, U. ~qARUSHIMA AND T. SENO, Biochim. Biophys. Acta, I42 (1967) 133. K. T*KEISHI, T. UKITA AND S. 2qlSHIMURA, J. Biol. Chem., 243 (1968) 5761. S. K. DUBE, K. A. MARCKER, B. F. C. CLARK AND S. CORY, Nature, 218 (1968) 232. B. F. C. CLARK, S. K. DUBE AND K. A. MARCKER, Nature, 219 (1968) 484 .
Received October 24th, 1968 Biochim. Biophys. Acta, 174 ( 1 9 6 9 ) 4 o 8 - 4 I I
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY NOTES BBA 91238
Restorotion of methionine-accepting ond tronsformylation octivity by combining oligonucleotide fragments derived from o ribonucleose TI digest of Escherichia coli t R N A fM°t Since the validity of the "cloverleaf" model 1 has been supported by investigations of a number of t R N A species whose nucleotide sequences have been established and since all of the physical and chemical evidence available is compatible with the cloverleaf arrangement 2, it would now be reasonable to discuss the nature of the t R N A molecule on the basis of this model. Using the 5'- and 3'-halves obtained by splitting the I - A bond of the presumed anti-codon, IAC of yeast t R N A val, BAYEV et al. 3 showed that the two halves restored the total valine-acceptor activity of the initial molecule only when mixed. The 5'and 3'-halves were obtained in our laboratory 4 from Escherichia coli t R N A wl, and they retained the valine-acceptor activity only when they were mixed and renatured. Preliminary data suggest that the cleavage occurred at the presumed anti-codon (unpublished results). This is good evidence that the aminoacyl-tRNA synthetase does not necessarily require the intactness of the anti-codon sequence in those t R N A molecules. As for the left-hand dihydrouridine loop, it could be a recognition site for the synthetase, since this loop is sufficiently different in all of the sequences determined so far 2. In this paper we show that t R N A ~Met from E . coli strain B could be split into two at the dihydrouridine loop region when the t R N A molecules were digested by ribonuclease T 1 in the presence of Mg *+ and that the resulting two halves restored the methionine-accepting and the transformylation ability when they were mixed. Thus the intactness of the dihydrouridine loop is not required either for the synthetase or the transformylase. The t R N A fMet was purified b y chromatography on a column of DEAE-Sephadex followed by chromatography on BD-cellulose, as reported previously 5 (crude t R N A was kindly prepared for us by Dr. S. ESUMI of Kaken Chemicals). 2o0 A260 m# units of the t R N A were dissolved in io ml of o.I M Tris-HC1 (pH 7.5) and 0.02 M magnesium acetate, and incubated at 37 ° for 26 rain together with 527 #g of ribonuclease T 1 (a kind gift of Dr. H. OKAZAKI of Central Research Laboratories, Sankyo Co.). The ribonuclease and hydrolysed products were removed from the partially digested t R N A by the method described previously 6. 97 A 26o m# units of the material were recovered. 14.5 % of the initial methionine-acceptor activity remained in this preparation which was heated in the presence of o.oi M EDTA (pH 7.o), 7 M urea and 0.02 M Tris-HC1 (pH 7.2) in a total volume of 3 ml at 80 ° for 4 rain, then rapidly cooled and subjected to a DEAE-Sephadex column chromatography at p H 2.7. As shown in Fig. I, the elution profile gave three peaks and one shoulder, which were named Fractions K, L, M and N, respectively. The materials were collected from each fraction by precipitation with 3.5 vol. of ethanol at --20 ° overnight followed b y centrifugation and dialysed against 0.05 M NaC1 and 0.02 M Tris-HC1 (pH 7.5) and then water to remove urea and salts. The methionine-accepting and the transformylation activities were assayed for each peak and combinations of t R N A fMet fragments Biochim. Biophys. Acta, 174 (1969) 4o8-411
409
PRELIMINARY N O T E S t
J
K L M
t
N
o
~0.08
L
25
o
510 F r a c t i o n No.
7"5 . . . .
- 1010
zL
'~
Fig. I. C h r o m a t o g r a p h i c s e p a r a t i o n of tRNAfMet f r a g m e n t s . 95 Azeo mu u n i t s of the ribonuclease Tl-treated tRNAfMet were applied to a D E A E - S e p h a d e x c o l u m n (0.5 cm × ioo cm) pre-equilib r a t e d w i t h 7 M urea solution (pH 2.7). The linear g r a d i e n t elution w a s carried o u t a t 20 ° using I5o ml of o.6 M NaC1 and 7 M u r e a (pH 2.7) in the reservoir a n d 15o ml of 7 M u r e a (pH 2.7) in the mixing vessel. The elution w a s complete w i t h i n 16 h. E a c h fraction contained 3 ml of the effluent. A b s o r b a n c e at 260 m/, ( 0 ) and at 330 m/* ( O ) was measured. The materials collected f r o m F r a c t i o n s 44-47, 49-52, 54-58 and 59-67 were designated F r a c t i o n s K, L, M and N, repectively.
TABLE I RESTORATION OF M E T H I O N I N E - A C C E P T O R
AND TRANSFORM'YLATION ACTIVITIES BY COMBINING
tRNAIMet FRAGMENTS O.O15 A2e0 mg u n i t of each f r a g m e n t as well as the i n t a c t t R N A was dissolved in a final v o l u m e of o.i ml of o.2 M NaC1, o.oi M MgC1 a and o.o2 M Tris-HC1 (pH 7.2) a n d i n c u b a t e d at 37 ° for i h. Methionine-acceptor 2 a n d the t r a n s f o r m y l a t i o n 8 activities were assayed as described previously.
Fragment
p4C]Methionine * accepted (counts/rain per o.o2 ml)
K L M N
5 8 133 lO 3
K+M K+lq L+M L+Iq M+lq
282 197 468
[14C] Formate** accepted
(counts/min per o.o4 ml) 2 o 37 28
Net increase
i334 516
144 89 327 i223 28o
I n t a e t R N A f M e t 1153
Net increase 56 52 122 360 I12
17 22
85 332 47
382
" Specific activity, 218 m C / m m o l e . ** Specific activity, 33 m C / m m o l e .
Biochim. Biophys. Acta, I74 (1969) 4 o 8 - 4 I I
410
PRELIMINARY NOTES
as presented in Table I. The results show that both activities were recovered to about 50 % of those of the native t R N A f~a only when the L and the N were mixed. Although not shown in Table I, the mixing of the K, L and M fractions did not give rise to the recovery, nor did the addition of the K and/or the M fraction to the mixture of the L and the N fractions inhibit the restoration. I t is interesting to know the nucleotide sequences of the oligonucleotide present in the L and N fractions. Since the complete nucleotide sequence of t R N A fM~t from E. coli has been proposed 9 and the column chromatographic elution pattern of the ribonuclease T,-digested t R N A ~iet is known (ref. 5 and unpublished data), 2 A2~0 m/, units each from Fractions L and N were subiected to chromatography to obtain the oligonucleotide compositions of the two fragments, as shown in Fig. 2. Although the L and N fractions are not very pure, 0
0025
0.015
4
= 0,005
.._/
E o
OFb
0.00
i'
0['-" %
o
100
200
300
400
500
Effluent vol. (ml)
Fig. 2. C h r o m a t o g r a p h y of a complete ribonuclease T x digest of tRNAiMet, the L f r a g m e n t and the N fragment. Samples were r u n on a D E A E - S e p h a d e x A-25 c o l u m n (o.55 cm × 15o cm) at n e u t r a l p H w i t h an NaC1 gradient f r o m o.14 to o. 7 M (4oo ml × 2) in the presence of 7 M urea, as described previously 5. (a) 3 A2e0 m~ u n i t s of t R N A f i e t , (b) 2 A2~0 m~ u n i t s of t h e L and (c) 2 A~6~ ma u n i t s of t h e N were used. The elution profile w a s o b t a i n e d in a cell w i t h a 4 - m m light p a t h b y a u t o m a t i c ultraviolet recorder of Model JLC-3BC ( J a p a n E l e c t r o n Optics L a b o r a t o r y Co.) w i t h a filter (~max, 260 m/g;dJtllZ, 7 m/~). The flow r a t e w a s 0.3 ml/min, mTG = 7-methylguanosine; t*U = 4-thiouridine; Cm = 2'-O-methylcytidine.
the following points could be seen from the comparison of the relative amounts of peaks obtained from the L and N fractions and the intact tRNA. The L and N fractions both possess one of the two tetranucleotides (Peak 6), since the L gave half the amount of Peak 6 given by intact tRNA, and the N fraction gave Peak 6 the amount of which is almost identical to Peak 2 which is comprised of two dinueleotides. These facts indicate that the cleavage occurred at the dihydrouridine region if judged on the basis of the t R N A fMet arranged in the cloverleaf pattern (see ref. 9). The following observations from the chromatograms are all compatible with the above judgement, leading to the conclusion that the L fraction represents the 3'half and the N the 5'-half of the tRNAfMet: (i) Many more Gp are in the N fraction. (2) Dinueleotides are not in the L but in the N fraction. (3) 4-Thioufidine is in the N fraction, as iudged b y its specific absorbance at 33o m#, shown in Fig. I (*U in Biochim. Bgophys. Acta, 174 (1969) 4o8-411
PRELIMINARY NOTES
411
the sequence established by DUBE et al2 was identified as 4-thiouridine: see ref. 5). (4) 7-Methylguanosine-containing tetranucleotide is in the L fraction. (5) Hexanucleotide-containing CCA terminus is in the L but not in the N fraction. (6) (Cp).~Gp and the two large fragments (Peaks 8 and 9) were in the L fraction. Detection of the minor components, including pCp, by the two-dimensional paper chromatography of the ribonuclease T 2 digest s of the L and N fractions also agreed with the above observations. Although not shown in Fig. I, the K fraction turned out to be the anti-codon loop fragment consisting of a nonadecanucleotide as reported by CLARK, DUBE AND MARCKER1° and the M fraction possessed all of the sequence of the L fraction except the hexanucleotide containing the CCA terminus. The maximum methionine-accepting activity was observed when the L and N fractions were mixed in a ratio of 2:1. This ratio agreed closely with the theoretical ratio, 2.3:1 , which was calculated with the aid of the molar extinction coefficient and the assumption that the sphtting occurled at -DpApGpCpUp- (D -- dihydrouridine) (see ref. 9). It should be mentioned that the restoration of those biological activities did not require particular conditions (temperature, salt concentration or pre-incubation period) just as for tRNA val from yeast z, while in the case of E. coli tRNA wl, the recovery depended on pre-incubation temperature, period and salt concentration 4. In conclusion, the L and N fractions are the 3'- and 5'-halves, respectively, of the tRNA fMet which result from splitting somewhere in the dihydromidine loop region. The methionine-accepting activity and the transformylation ability were retained only when the two halves were mixed. Determination of the precise site of the break in the dihydrouridine loop region and further study on the other biological properties of the fragments are now in progress.
Virology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)
TAKESHI SENO MASARU KOBAYASHI SusoMu NISHIMURA
I R. J. 2 J. 3 A. 4 5 6 7 8 9 IO
W. HOLLEY, J. APGAR, G. A. EVERETT, J. T. MADISON, M. MARQUISEE, S. H. MERRILL, R. PENSWICK AND A. ZAMIR, Science, 147 (1965) 1462. T. MADISON, Ann. Rev. Biochem., 37 (1968) 131. A. BAYEV, I. FODOR, A. D. MIRZABEKOV, V. D. AXELROD AND L. Y. KAZARINOVA, Molekularnaya Biologiya, I (1967) 859. K. ODA, F. KIMURA, F. HARADA AND S. NISHIMURA, Biochim. Biophys. Acta, in t h e press. T. SENO, M. KOBAYASHI AND S. NISHIMURA, Biochim. Biophys. Acta, 169 (1968) 80. S. NISHIMURA AND G. D. ~OVELLI, Proc. Natl. Acad. Sci. U.S., 53 (1965) 178. S. lX]'ISHIMURA, F. HARADA, U. ~qARUSHIMA AND T. SENO, Biochim. Biophys. Acta, I42 (1967) 133. K. T*KEISHI, T. UKITA AND S. 2qlSHIMURA, J. Biol. Chem., 243 (1968) 5761. S. K. DUBE, K. A. MARCKER, B. F. C. CLARK AND S. CORY, Nature, 218 (1968) 232. B. F. C. CLARK, S. K. DUBE AND K. A. MARCKER, Nature, 219 (1968) 484 .
Received October 24th, 1968 Biochim. Biophys. Acta, 174 ( 1 9 6 9 ) 4 o 8 - 4 I I