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Restoration of valine acceptor activity by combining oligonucleotide fragments derived from a Bacillus subtilis ribonuclease digest of Escherichia coli valine transfer RNA
97
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96142
R E S T O R A T I O N OF V A L I N E ACCEPTOR A C T I V I T Y BY COMBINING O L I G O N U C L E O T I D E FRAGMENTS D E R I V E D FROM A B A C I L L U S S U B T I L I S R I B O N U C L E A S E D I G E S T OF E S C H E R I C H I A COLI V A L I N E T R A N S F E R RNA
K I N I C H I R O ODA, F U M I K O K I M U R A , F U M I O H A R A D A AND S U S U M U N 1 S H I M U R A
Virology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan) (Received O c t o b e r 4th, I968)
SUMMARY
Valine tRNA from Escherichia coli, treated with Bacillus subtilis ribonuclease in the presence of Mg ~+ yielded two large fragments. They contained the - C - C - A end and the pGp end, respectively, and could be separated from each other by means of DEAE-cellulose column chromatography followed by rechromatography with D E A E Sephadex A-25 at p H 2.7. When the two fragments were combined and renatured in the presence of Mg *+, valine acceptor activity was fully restored. It is essential to combine the two fragments for the reactivation of valine acceptor activity. No acceptor activity was restored with either component alone. The restoration of valine acceptor activity was temperature and time dependent, and the presence of divalent ion such as Mg 2+ was also necessary. The thermal denaturation of the ribonuclease-treated valine tRNA occurred at quite a lower temperature than that of native valine tRNA. From the data of thermal denaturation profile of the two fragments and their reconstituted molecule, a possible mechanism for the renaturation process will be discussed.
INTRODUCTION
t R N A can be altered by treatment with Bacillus subtilis ribonuclease so as to retain its ability to accept amino acids, but not to transfer amino acids into protein 1-~. The acceptor activities, however, are inactivated by means of heat denaturation s. These observations, obtained with unfractionated tRNA, led us to study more precisely the unique property of the enzymatically altered tRNA with purified tRNA 4. The present communication indicates that valine t R N A from Escherichia coli B, treated with ribonuclease from B. subtilis, yields two large fragments (containing the - C - C - A end and the pGp end, respectively) that can be separated and that when combined and 'renaturated' in the presence of Mg 2+, regain valine-accepting ability. The kinetics of the restoration of valine acceptor activity of the t R N A fragments are also presented. Biochim. Biophys. Acta, 179 (1969) 97-1o5
98
K. ODA et al.
BAYEV et al. 5 previously reported a similar observation with a yeast valine tRNA. They have demonstrated that partial cleavage of yeast valine t R N A b y guanyloribonuclease results in a single cleavage of the tRNA. The two fragments thus produced do not accept valine, but when they are mixed, the aggregate formed can be acylated with valine. Thus our results reported here are fully compatible with their observations, although the source of valine t R N A used is different from yeast. MATERIALS AND METHODS
Preparation oj valine t R N A ]rom E. coli B Three column chromatographic procedures were used successively to obtain valine tRNA, The first and second were carried out as described previously 4. Finally, benzoylated DEAE-cellulose column chromatography, as described by GILLAM et al. 6, was adopted to remove other tRNA's from valine tRNA. Valine t R N A thus obtained could accept 1.6 nmoles of [14C]valine per A unit* of the tRNA, and the chromatography of ribonuclease T 1 digest of valine t R N A indicated that it was pure. This valine t R N A was recognized mainly by G - U - A and G - U - G , and less well by G--U-U when tested in tRNA-ribosome binding experiments 7.
Preparation o[ ribonuelease-treated valine t R N A In order to determine the optimal concentration of B. subtilis ribonuclease required to obtain modified valine tRNA, E. coli valine t R N A was treated with an increasing concentration of B. subtilis ribonuclease and diluted 4o-fold with cold distilled water. Aliquots of the samples, with or without heating and rapid cooling, were assayed for valine-acceptor activity. The activity without heating gradually decreased with increasing amounts of B. subtilis ribonuclea~e: hcwever, at a concentration of 4 units**/ml of the ribonuclease per 25 A units of valine tRNA/ml, 85%of the activity remained, but only 6 % of it remained after heating. This condition was used for the preparation of the treated valine tRNA. A typical example is as follows: 320 A units of valine t R N A dissolved in 20 ml of o.i M Tris-HC1 (pH 7.5) and 0.02 M magnesium acetate was treated with 50 units of B. subtilis ribonuclease at 37 ° for 13 rain. After the incubation, the reaction mixture was cooled in an ice-water bath and loaded on a column of DEAE-cellulose (i cm × 5 cm) previously equilibrated at 4 ° with o.I M NaC1, o.oi M MgCI., and 0.02 M Tris-HC1 (pH 7.5). The column was washed with 7 ° m l of 0.2 M NaCI, o.oi M MgC12 and 0.o2 M Tris-HC1 (pH 7.5) precipitated by adding 2.5 voh of ethanol, and dissolved in 2 ml of cold o.ooi M MgC12 and 0,02 M Tris-HC1 (pH 7.5)-
Separation o[ valine t R N A [ragments To the treated valine t R N A (12o A units in 2.2 ml), o.I vol. of o.i M E D T A (pH 7.0) was added. The mixture was heated at 9 °0 for 4 rain and quickly cooled in " A u n i t s r e f e r t o a b s o r b a n c e m e a s u r e m e n t in i m l v o l u m e a t 260 m/z a n d a t n e u t r a l p H using a l-cm light-path quartz cuvette. ** i u n i t of r i b o n u c l e a s e in t h i s r e p o r t is t h e a m o u n t of r i b o n u c l e a s e a c t i v i t y c o m p a r a b l e t o /~g o f b o v i n e p a n c r e a t i c r i b o n u c I e a s e w i t h y e a s t r i b o s o m a l R N A a s a s u b s t r a t e .
Biochim, Biophys. Acta, i 7 9 (1969) 9 7 - 1 o 5
ACCEPTOR ACTIVITY OF VALINE
tRNA
FRAGMENTS
99
an ice-water bath. Solid urea was added to the solution to give a final concentration of 7 M. The sample was loaded onto a column of DEAE-cellulose (Whatman D E 23: column size, 0.5 cm × 15o cm). Elution was performed with a linear gradient of NaC1 as described b y PENSWICK AND HOLLEy8: The mixing chamber contained 400 ml of 7 M urea, 0.02 M Tris-HC1 (pH 8.0) and 0.25 M NaC1, and the reservoir contained 400 ml of 7 M urea, 0.02 M Tris-HC1 buffer (pH 8.0) and 0.65 M NaC1. The flow rate was 6 ml/h. Fractions of 2.7 ml were collected.
Reehromatography o] the ]ragments The valine t R N A fragments obtained by the first chromatography were precipitated by the addition of 3.5 vol. of ethanol. The mixture was allowed to stand overnight at --20 °. The fragments were collected by centrifugation at io ooo rev./min for IO rain, and dissolved in 1.5 ml of 7 M urea. Rechromatography of the fragments at p H 2. 7 was performed by the use of a column of D E A E - S e p h a d e x A-25 (0.5 cm × 50 cm) as described by RUSHIZKY et al2. Elution was carried out with a linear gradient of NaC1, the mixing chamber containing IOO ml of 7 M urea and 0.06 M HC1, and the reservoir containing IOO ml of 7 M urea, 0.06 M HC1 and 0.6 M NaC1. The flow rate was 20 ml/h. Fractions of 2.7 ml were collected. Each fraction obtained was diluted 5 fold with 0.02 M Tris-HC1 buffer (pH 7.5) loaded on a column of DEAEcellulose (I cm × 5 cm), washed with 50 ml of 0.02 M Tris-HC1 buffer (pH 7.5) and eluted with 20 ml of I M NaC1 and 0.02 M Tris-HC1 buffer (pH 7.5). Other miscellaneous procedures Aminoacyl-tRNA synthetases from E. coli B were prepared as described previously 4. The conditions for the assay of valine-acceptor activity were the same as those previously reported 4. The procedure for the preparation of E14CIvalyl-tRNA has also been described previously 1°. Assay of aminoacyl-tRNA binding to ribosomes was carried out b y the procedure described b y NIRENBERG AND LEDER7. The reaction mixture (0.05 rot) contained o.I M Tris-HC1 buffer (pH 7.5) 0.05 M KC1, I A unit of ribosomes, 0.02 M magnesium acetate, EI*Clvalyl-tRNA and trinucleoside diphosphate as specified. Incubation was carried out at 25 ° for 15 rain. Materials B. subtilis ribonuclease was the product reported previouslyll,lL Ribonuclease T1 and T~ were kindly donated b y Dr. H. Okazaki of Central Research Laboratories, Sankyo. G - U - A and G - U - G were the gifts of Dr. T. Ukita, University of Tokyo. Uniformly labeled E14Clvaline (specific activity, 208.5 #C/#mole) was obtained from New England Nuclear. W h a t m a n D E 23 (capacity, I mequiv/g) was obtained from Balston. DEAE-Sephadex A-25 (capacity, 3.5±0.5 mequiv/g; particle size, 4o-12o #) was a product of Pharmacia Fine Chemicals. RESULTS
Inability o] B. subtilis ribonuclease-treated valyl-tRNA to bind to ribosomes As seen in Table I, the ability of B. subtilis ribonuclease-treated valyl-tRNA to bind to ribosomes in the presence of G - U - A and G - U - G was very little when comBiochim. Biophys. Acta. 179 (1969) 97-1o5
ioo
i~. O D A
et al.
TABLE I INABILITY
OF
B. subtilis
RIBONUCLEASE-TREATED
VALYL-tRNA
TO B I N D TO R I B O S O M E S
The reaction m i x t u r e contained 7700 counts/rain of [14C]valyl-tRNA. 6090 c o u n t s / m i n of B. subtilis ribonuclease-treated [i4C]valyl-tRNA, 0.o 5 A u n i t of G - U - A and 0.05 A unit of G - U - G where indicated.
Template
[14C] Valyl-tRNA (counts/min bound to ribosomes)
B. subtilis ribonuclease-treated [14Cjvalyl-tRNA (counts~rain bound to ribosomes)
None G-U-A G-U-G
128 1536 247 °
193 265 297
pared with that of untreated control i14Clvalyl-tRNA. Although not shown in the table, lack of activity of the treated valyl-tRNA in a cell-free protein-synthesizing system directed by poly (Ua,G) was also found, as previously reported with unfractionated t R N A preparations 3.
Restoration of valine acceptor activity of the denatured valine tRNA by renaturation The valine acceptor activity of the ribonuclease-treated valine tRNA was completely inactivated after heating and rapid cooling as shown in Table II. This is in accordance with the previous observation obtained with unfractionated t R N A 3. However, the activity was found to be recovered markedly when the heated t R N A was slowly cooled (20 h) from 85 ° to 3 °0 in a thermos flask. TABLE II RESTORATION
OF VALINE
ACCEPTOR ACTIVITY OF TIlE RIBONUCLEASE-TREATED
VALINE
tRNA
BY
SLOW COOLING
The t R N A (0.o 5 A unit) was dissolved in 0.2 ml of o.2 M NaC1, 0.02 M Tris-HC1 buffer (pH 7.2) and O.Ol M MgCI,, and heated at 85 ° for 5 min. I n the case of rapid cooling, the sample was p u t in an i c e - w a t e r bath.
B. subtilis ribonuclease-treated valine t R N A
Native valine t R N A
[x4ClValine a t t a c h e d (counts/min/o.o 5 ml) Per cent of activity
Control
Heat and rapid cool
Heat and slow cool
Control
Heat and rapid cool
Heat and slow cool
5395 IOO
4946 91. 5
4843 89.7
3287 IOO
179 5.4
216o 65.5
Separation o] valine tRNA ]ragments by DEAE-cellulose column chromatography The ribonuclease-treated valine t R N A was heat-denatured as described in MATERIALS AND METHODS and chromatographed by the use of DEAE-cellulose. The profile of the eluted valine t R N A fragments is shown in Fig. Ia. The two main peaks (II and III) composed of the - C - C - A and the pGp halves of the valine t R N A molecule, respectively, were purified b y rechromatography as shown in Figs. I b and ic. A1Biochim. Biophys. Acta, 179 (1969) 97-1o5
ACCEPTOR ACTIVITY OF VALINE I II I II J
I
Ill
60
80
tRNA II
IV
FRAGMENTS
I0I
I
1.0 t
'4~
1
"
30
40
@
~
20
40
50
70
90
100 110
,
60
80 20 Fraction number
40
60
80
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 valine t R N A f r a g m e n t s , a. S e p a r a t i o n of v a l i n e - t R N A f r a g m e n t s b y D E A E - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y , b. R e c h r o m a t o g r a p h y of F r a g m e n t II b y D E A E - S e p h a d e x A-25 c o l u m n c h r o m a t o g r a p h y , c. R e c h r o m a t o g r a p h y of F r a g m e n t I I I b y D E A E - S e p h a d e x A-25 c o l u m n c h r o m a t o g r a p h y . F o r detail, see MATERIALS AND METHODS.
though not shown in Fig. I, Peaks I and IV were also purified by rechromatography. Peak I consisted of two fragments (Ia and Ib) which were found to be the fragments derived from Component II by further cleavage according to the pattern of the chromatography of ribonuclease T1 digestion products. Peak IV also consisted of two fragments. Fragment IVa, which included two thirds of the total amount of Peak IV, was somewhat similar to Fragment II, and Fragment IVb was found to be the same as Fragment III. These were also suggested by analogy with the products of ribonuclease T 1 digestion. TABLE III RESTORATION OF VALINE ACCEPTOR ACTIVITY BY COMBINING VALINE t R N A FRAGMENTS T h e f r a g m e n t s as specified were dissolved in 0.2 ml of 0.2 M NaC1, 0.02 M Tris-HC1 b u f f e r (pH 7.2) a n d 0.oi M MgCI~. Expt. No.
Addition o[ the lragment Ia A units added/o.2 rot: 0.o3
I
2 3 4 5 6 7 8 9 IO II
Fl*C]Valine accepted (countslmin/o.o5 ml)
lb
II
III
IVa
0.02
o.o97
o.io8
o.o54
+
o
+
6 +
o
+ + + +
Heat and slow cool
+
+ + + N a t i v e valine t R N A , 0.03 A u n i t N a t i v e valine t R N A , 0.06 A u n i t
+ + + +
+
io o 3169 87 1734 3325
No treatment
o
o 2 2 o 128 5 IOO 168 2609 4572
Biockim. Biophys. Acta, 179 (1969) 97 lO5
K. ODA et al.
lO2
Restoration o[ valine acceptor activity ~, combining the [ragments When each fragment was heated and separately cooled slowly (No. i-5) , no valine acceptor activity was restored at all as shown in Table h i . However, surprisingly, when Fragments I I and I I I were mixed and slowly cooled (No. 6), valine acceptor activity was recovered to the extent of approx. 73 % compared with the same A units of native untreated valine tRNA (No. io, Ii). Fragment II could not be replaced by Fragment Ia and Ib, produced by the further cleavage of Fragment I I (No. 7). Furthermore, addition of Fragments Ia and Ib to the mixture of Fragments II and I I I did not alter the extent of the restoration significantly (No. 9). Fragment IVa, which was found to be similar to Fragment n, could be substituted for Fragment I I (No. 8). It should be mentioned that the restoration of activity was very poor when the mixture was assayed without the renaturation procedure.
Temperature dependence o[ the renaturation process ]'he mixture of Fragments II and n I was incubated at 85, 65 and 5°o and slowly cooled to 25 ° in 20 h in each case. The valine acceptor activity was restored to the same extent in every case (7° % as compared with control valine tRNA). In addition it was found that incubation of the mixture at 37 ° for 20 h was enough for maximal restoration of valine acceptor activity. As shown in Fig. 2, the rate of the reactivation was measured by incubating the mixture at different temperatures. The reactivation occurred even at 25 °, but proceeded very slowly. At 37 °, the reactivation proceeded much faster than at 25 ° , and the maximal reactivation was attained after incubation for 2o h. It should be mentioned that when Fragments II and I I I were separately incubated at 37 ° for 2o h, and then mixed followed by innnediate assay, no valine acceptor activity was restored. ! u~
d
i
cl c
6O0
s 4001 o
'~'
500, /i
~ 2 5 °
tt~' /
20oI ;0 '
Time
h
)
i0 '
:0
Concentration of M £ -
lO "1
Fig. 2. Time course of the r e s t o r a t i o n of valine acceptor activity b y reconstitution of F r a g m e n t s I I and I I I . F r a g m e n t s I I and 11I were mixed (o.33 and 0.48 A u n i t / m l respectively) ill 0.2 M NaC1, 0.02 M Tris-HC1 (pH 7.2) and o.o~ M MgC12, and incubated at 37 or 25 °. At the times indicated, aliquots were removed, rapidly cooled and stored at --20 °. Valine acceptor activities were assayed as described previously 4 except t h a t the time of incubation was 5 rain. Fig. 3. R e q u i r e m e n t of Mg ~+ for the r e s t o r a t i o n of valine acceptor activity. F r a g m e n t I I and I I I (0.03 A u n i t each) were mixed in 0.2 ml of 0.2 M NaCI, 0.02 M Tris-HC1 buffer (pH 7,2) and MgClz as specified. I n c u b a t i o n was at 37 ° for 20 h.
Biochim. Biophys. Acta, 179 (1969) 97-1o5
ACCEPTOR ACTIVITY OF VALINE
tRNA
103
FRAGMENTS
Ellect ol magnesium concentration on the restoration o/valine acceptor activity As shown in Fig. 3, the restoration of valine acceptor activity depends on the presence of Mg 2÷. It did not occur at concentrations of Mgz÷ lower than o.ooi M, and the maximal restoration was found at 0.025 M.
E/leer o/ relative concentration o/ Fragments I I and I I I on the restoration o/ valine acceptor activit# As shown in Fig. 4, constant amounts of Fragment I I (or III) were incubated at 37 ° for 20 h with increasing amounts of Fragment I I I (or II), and their acceptor activities were then measured. In both cases, the extent of the restoration of activity increased linearly until the same amount of the complementary fragment was added, and after the addition of a 3-fold excess of the complementary fragment, the increase of activity levelled off. A 14 °/o higher plateau level was attained with Fragment I I I . This m a y indicate that either (i) the length of Fragment I I I is slightly less than that of Fragment II, or (2) Fragment I I I has a greater affinity for Fragment II. a 0"61
. 800 o b
'
~'"
/mr
Native vali
~,f
i
f ~ - ~ ~ i ~ % ~ setPeclted valine t R N A
ff
/mL
"~ 600
o,4--
I
30
40
50
60
70
I
80
90
b
~-~m 4001 ff
2ooy ~
~ ." :t. ~
i
0.2 0.4 0.6 0.8 Amount of complementary fragment (A units/m/.)
~
i
:
~
{I
O.7 ! 30 °
40 °
50 ° 60 o Temperature
70 °
80 °
9() °
Fig. 4. E f f e c t of r e l a t i v e c o n c e n t r a t i o n of F r a g m e n t s I I a n d I I I on t h e r e s t o r a t i o n of v a l i n e acc e p t o r a c t i v i t y . A c o n s t a n t a m o u n t of F r a g m e n t I I or I I I (0.03 A u n i t ) w a s m i x e d w i t h i n c r e a s i n g a m o u n t s of F r a g m e n t I I I or I I r e s p e c t i v e l y in 0.2 ml of 0.2 M NaC1, 0.02 M Tris-HC1 buffer (pH 7.2) a n d o.o i M MgC1 v I n c u b a t i o n w a s a t 37 ° for 20 h. Fig. 5- The t h e r m a l d e n a t u r a t i o n c u r v e of n a t i v e v a l i n e t R N A , B. subtilis r i b o n u c l e a s e - t r e a t e d v a l i n e t R N A a n d i t s { r a g m e n t s in t h e p r e s e n c e of Mg 2+. A b s o r b a n c e a t 26o mt~ w a s m e a s u r e d i n 0.2 M NaC1, 0.02 M T r i s - H C l buffer (pH 7.2) a n d o.oi M MgC1 v The f r a g m e n t s w e re i n c u b a t e d a t 37 ° for 2o h before t h e h e a t t r e a t m e n t was begun. In t h e case of t h e m i x t u r e of F r a g m e n t I I a n d III, t h e s a m p l e c o n t a i n e d 0.33 A u n i t of F r a g m e n t I I a n d 0.36 A u n i t of F r a g m e n t I I I .
Thermal denaturation o / B . subtilis ribonuclease-treated valine tRNA and its/ragments As shown in Fig. 5a, thermal denaturation of the ribonuclease-treated valine tRNA occurred at a lower temperature than that of control native valine tRNA. Biochim. Biophys. Acta, 179 (1969) 97-1o5
lO4
K. ODA et al.
Fig. 5 b shows the thermal denaturation profile of Fragment II, Fragment I I I and their reconstituted molecule. The pattern of the curve of the reconstituted molecule is quite similar to that of B. subtilis ribonuclease-treated valine tRNA, although the percent increase of absorbance of the reconstituted molecule was not as high as that of the ribonuclease-treated valine tRNA. It should be mentioned that Fragment I I or Fragment I I I alone gave distinctive denaturation profiles, and the theoretical curve obtained by taking the mean value of their patterns was not quite the same as that of the reconstituted molecule. In addition, when Fragments I I and I I I were mixed to form the reconstituted molecule, no decrease of absorbance at 260 m,u was detected even after incubation at 37 ° for 20 h. DISCUSSION
The results reported above clearly indicate that valine acceptor activity is completely restored by combining two large fragments (Fragment II, containing the - C - C - A end and Fragment I I I , containing the pGp end) which were obtained b y tLe limited B. subtilis ribonuclease digestion of E. coli valine tRNA. It is essential to combine the two fragments for the reactivation of valine acceptor activity. No acce Ftor activity was restored with either component alone. Furthermore, the reappearance of valine-acceptor activity is temperature and time dependent, and the presence of Mg 2+ is also necessary. Although not shown in RESULTS, it should be mentioned that other divalent cations such as Ca 2+ and Mn 2+ are also active as well as Mg ~+. Furthermore, if the concentration of NaC1 during the renaturation process is increased up to 0.8 M, valine acceptor activity is fully restored even in the absence of the divalent ions (S. NISHIMURA, unpublished data). The association of the two fragments, which is required to accept the amino acid, must take place gradually during the renaturation process. However, no decrease of absorbance at 260 m# was observed during the course of renaturation. This suggests that either (i) interaction of the two fragments that contributes to tile decrease of absorbance occurs in a very small fraction or region as compared with the total molecule, or (2) conformational rearrangements can take place without changing the gross amount of secondary and tertiary structure. At any rate, it should be emphasized that renaturation of this type is a unique reaction since the fragments are composed of relatively short chains of oligonucleotides and the extent of the renaturation is expressed as specific biological activity. The fragments (II and III) are oligonueleotides derived from the - C - C - A and pGp end of the t R N A molecule, respectively. T.be two fragments are approximately the same size, and include most of the sequence of native valine tRNA. This was suggested by comparison of the chromatographic patterns of the ribonuclease T 1 digest of the fragments with that of native valine tRNA. In addition, two-dimensional paper chromatography of a ribonuclease T 2 digest of the fragments 4 showed that Fragment I I contained I mole each of adenosine, N7-methylguanylic acid, N6-methyladenylic acid*, ribothymidylic acid and pseudouridylic acid, and Fragment I I I contained i * W i t h o u t a l k a l i n e t r e a t m e n t , I m o l e of N n - m e t h y l a d e n y l i c a c i d w a s f o u n d i n E. coli v a l i n e t R N A . S i n c e i t is n o t d e r i v e d b y t h e c o n v e r s i o n of N l - m e t h y l a d e n y l i c a c i d o r N S - i s o p e n y e n y l a d e n y l i c a c i d 14,1~, i t is a n e w m i n o r b a s e c o m p o n e n t f o u n d i n t R N A . D e t a i l s of t h i s w i l l b e p u b lished elsewhere.
Biochim. Biophys. Acta, 179 (I969) 9 7 - 1 o 5
ACCEPTOR ACTIVITY OF VALINE
tRNA
FRAGMENTS
Io 5
mole each of guanosine 3',5'-diphosphate and 4-thiouridylic acid. Thus all minor components detected in valise t R N A so far are present in either Fragment II or Fragment III. However, these results do not necessarily mean that two fragments are produced by a single cleavage of valine-tRNA molecule as described by PENSWlCK AND HOLLEY8 and BAYEV et al. 13, although it is most probable case. To clarify this point, a sequential analysis of E. coli valine t R N A is now under investigation. ACKNOWLEDGMENTS
We thank Dr. Sinichiro Esumi, Laboratories of Kaken Chemicals for the largescale isolation of crude E. coli tRNA. The authors are indebted to Dr. G. David Novelli for the critical reading and revision of the manuscript. The work was supported partly by research grant No. 94216, 954025 and 954060 from Ministry of Education. We thank Dr. Dieter $611, Yale University for informing us that better fractionation of E. coli t R N A on benzoylated DEAE-cellulose columns can be achieved at 4 ° .
REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15
S. NISHIMURA AND G. D. NOVELLI, Biochem. Biophys. Res. Commun., i i (1963) i 6 i . S. NISHIMURA AND G. D. NOVELLI, Biochim. Biophys. Acta, 80 (1964) 574. S. NISHIMURA AND G. D. NOVELLI, Proc. Natl. Acad. Sci. U.S., 53 (1965) 178. S. NISHIMURA, F. HARADA, U. NARUSHIMA AND T. SENO, Biochim. Biophys. Acta, 142 (1967) 133. A. A. BAYEV, [. FODOR, A. D. MIRZABEKOV, V. D. AXELROD AND L. Y. KAZARINOVA, Molekularnaya Biologiya, i (1967) 859. I. GILLAM, S. MILLWARD, D. BLEW, M. VON TIGERSTROM, E. WIMMER AND G. M. TENNER, Biochemistry, 6 (1967) 3043 . M. W. NIRENBERG AND P. LEDER, Science, 145 (1964) 1399. J. R. PENSWICK AND R. W. HOLLEY, Proc. Natl. Acad. Sci. U.S., 53 (1965) 543. G. W. RUSHIZKY, E. M. BARTOS AND H. A. SOBER, Biochemistry, 3 (1964) 626. S. ~qlSHIMURA, F. HARADA AND M. HIRABAYASHI, J. Mol. Biol., i n the press. S. NISHIMURA, Biochim. Biophys. Acta, 45 (196o) 15. S. NISHIMURA AND H. OZAWA, Biochim. Biophys. Acta, 55 (1962) 421. A. A. BAYEV, A. n . MIRZABEKOV, V. D. AXELROD, T. V. VENKSTERN, L. LI, A. I. KRUTILINA, I. EODOR AND L. Y. KAZARINOVA, Dokl. Akad. Nauk SSSR, 173 (1967) 204. R. H. HALL, Biochemistry, 4 (1965) 661. Y. IWANAMI AND G. M. BROWN, Arch. Biochem. Biophys., 124 (1968) 472.
Biochim. Biophys. Acta, 179 (1969) 97-1o5
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96142
R E S T O R A T I O N OF V A L I N E ACCEPTOR A C T I V I T Y BY COMBINING O L I G O N U C L E O T I D E FRAGMENTS D E R I V E D FROM A B A C I L L U S S U B T I L I S R I B O N U C L E A S E D I G E S T OF E S C H E R I C H I A COLI V A L I N E T R A N S F E R RNA
K I N I C H I R O ODA, F U M I K O K I M U R A , F U M I O H A R A D A AND S U S U M U N 1 S H I M U R A
Virology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan) (Received O c t o b e r 4th, I968)
SUMMARY
Valine tRNA from Escherichia coli, treated with Bacillus subtilis ribonuclease in the presence of Mg ~+ yielded two large fragments. They contained the - C - C - A end and the pGp end, respectively, and could be separated from each other by means of DEAE-cellulose column chromatography followed by rechromatography with D E A E Sephadex A-25 at p H 2.7. When the two fragments were combined and renatured in the presence of Mg *+, valine acceptor activity was fully restored. It is essential to combine the two fragments for the reactivation of valine acceptor activity. No acceptor activity was restored with either component alone. The restoration of valine acceptor activity was temperature and time dependent, and the presence of divalent ion such as Mg 2+ was also necessary. The thermal denaturation of the ribonuclease-treated valine tRNA occurred at quite a lower temperature than that of native valine tRNA. From the data of thermal denaturation profile of the two fragments and their reconstituted molecule, a possible mechanism for the renaturation process will be discussed.
INTRODUCTION
t R N A can be altered by treatment with Bacillus subtilis ribonuclease so as to retain its ability to accept amino acids, but not to transfer amino acids into protein 1-~. The acceptor activities, however, are inactivated by means of heat denaturation s. These observations, obtained with unfractionated tRNA, led us to study more precisely the unique property of the enzymatically altered tRNA with purified tRNA 4. The present communication indicates that valine t R N A from Escherichia coli B, treated with ribonuclease from B. subtilis, yields two large fragments (containing the - C - C - A end and the pGp end, respectively) that can be separated and that when combined and 'renaturated' in the presence of Mg 2+, regain valine-accepting ability. The kinetics of the restoration of valine acceptor activity of the t R N A fragments are also presented. Biochim. Biophys. Acta, 179 (1969) 97-1o5
98
K. ODA et al.
BAYEV et al. 5 previously reported a similar observation with a yeast valine tRNA. They have demonstrated that partial cleavage of yeast valine t R N A b y guanyloribonuclease results in a single cleavage of the tRNA. The two fragments thus produced do not accept valine, but when they are mixed, the aggregate formed can be acylated with valine. Thus our results reported here are fully compatible with their observations, although the source of valine t R N A used is different from yeast. MATERIALS AND METHODS
Preparation oj valine t R N A ]rom E. coli B Three column chromatographic procedures were used successively to obtain valine tRNA, The first and second were carried out as described previously 4. Finally, benzoylated DEAE-cellulose column chromatography, as described by GILLAM et al. 6, was adopted to remove other tRNA's from valine tRNA. Valine t R N A thus obtained could accept 1.6 nmoles of [14C]valine per A unit* of the tRNA, and the chromatography of ribonuclease T 1 digest of valine t R N A indicated that it was pure. This valine t R N A was recognized mainly by G - U - A and G - U - G , and less well by G--U-U when tested in tRNA-ribosome binding experiments 7.
Preparation o[ ribonuelease-treated valine t R N A In order to determine the optimal concentration of B. subtilis ribonuclease required to obtain modified valine tRNA, E. coli valine t R N A was treated with an increasing concentration of B. subtilis ribonuclease and diluted 4o-fold with cold distilled water. Aliquots of the samples, with or without heating and rapid cooling, were assayed for valine-acceptor activity. The activity without heating gradually decreased with increasing amounts of B. subtilis ribonuclea~e: hcwever, at a concentration of 4 units**/ml of the ribonuclease per 25 A units of valine tRNA/ml, 85%of the activity remained, but only 6 % of it remained after heating. This condition was used for the preparation of the treated valine tRNA. A typical example is as follows: 320 A units of valine t R N A dissolved in 20 ml of o.i M Tris-HC1 (pH 7.5) and 0.02 M magnesium acetate was treated with 50 units of B. subtilis ribonuclease at 37 ° for 13 rain. After the incubation, the reaction mixture was cooled in an ice-water bath and loaded on a column of DEAE-cellulose (i cm × 5 cm) previously equilibrated at 4 ° with o.I M NaC1, o.oi M MgCI., and 0.02 M Tris-HC1 (pH 7.5). The column was washed with 7 ° m l of 0.2 M NaCI, o.oi M MgC12 and 0.o2 M Tris-HC1 (pH 7.5) precipitated by adding 2.5 voh of ethanol, and dissolved in 2 ml of cold o.ooi M MgC12 and 0,02 M Tris-HC1 (pH 7.5)-
Separation o[ valine t R N A [ragments To the treated valine t R N A (12o A units in 2.2 ml), o.I vol. of o.i M E D T A (pH 7.0) was added. The mixture was heated at 9 °0 for 4 rain and quickly cooled in " A u n i t s r e f e r t o a b s o r b a n c e m e a s u r e m e n t in i m l v o l u m e a t 260 m/z a n d a t n e u t r a l p H using a l-cm light-path quartz cuvette. ** i u n i t of r i b o n u c l e a s e in t h i s r e p o r t is t h e a m o u n t of r i b o n u c l e a s e a c t i v i t y c o m p a r a b l e t o /~g o f b o v i n e p a n c r e a t i c r i b o n u c I e a s e w i t h y e a s t r i b o s o m a l R N A a s a s u b s t r a t e .
Biochim, Biophys. Acta, i 7 9 (1969) 9 7 - 1 o 5
ACCEPTOR ACTIVITY OF VALINE
tRNA
FRAGMENTS
99
an ice-water bath. Solid urea was added to the solution to give a final concentration of 7 M. The sample was loaded onto a column of DEAE-cellulose (Whatman D E 23: column size, 0.5 cm × 15o cm). Elution was performed with a linear gradient of NaC1 as described b y PENSWICK AND HOLLEy8: The mixing chamber contained 400 ml of 7 M urea, 0.02 M Tris-HC1 (pH 8.0) and 0.25 M NaC1, and the reservoir contained 400 ml of 7 M urea, 0.02 M Tris-HC1 buffer (pH 8.0) and 0.65 M NaC1. The flow rate was 6 ml/h. Fractions of 2.7 ml were collected.
Reehromatography o] the ]ragments The valine t R N A fragments obtained by the first chromatography were precipitated by the addition of 3.5 vol. of ethanol. The mixture was allowed to stand overnight at --20 °. The fragments were collected by centrifugation at io ooo rev./min for IO rain, and dissolved in 1.5 ml of 7 M urea. Rechromatography of the fragments at p H 2. 7 was performed by the use of a column of D E A E - S e p h a d e x A-25 (0.5 cm × 50 cm) as described by RUSHIZKY et al2. Elution was carried out with a linear gradient of NaC1, the mixing chamber containing IOO ml of 7 M urea and 0.06 M HC1, and the reservoir containing IOO ml of 7 M urea, 0.06 M HC1 and 0.6 M NaC1. The flow rate was 20 ml/h. Fractions of 2.7 ml were collected. Each fraction obtained was diluted 5 fold with 0.02 M Tris-HC1 buffer (pH 7.5) loaded on a column of DEAEcellulose (I cm × 5 cm), washed with 50 ml of 0.02 M Tris-HC1 buffer (pH 7.5) and eluted with 20 ml of I M NaC1 and 0.02 M Tris-HC1 buffer (pH 7.5). Other miscellaneous procedures Aminoacyl-tRNA synthetases from E. coli B were prepared as described previously 4. The conditions for the assay of valine-acceptor activity were the same as those previously reported 4. The procedure for the preparation of E14CIvalyl-tRNA has also been described previously 1°. Assay of aminoacyl-tRNA binding to ribosomes was carried out b y the procedure described b y NIRENBERG AND LEDER7. The reaction mixture (0.05 rot) contained o.I M Tris-HC1 buffer (pH 7.5) 0.05 M KC1, I A unit of ribosomes, 0.02 M magnesium acetate, EI*Clvalyl-tRNA and trinucleoside diphosphate as specified. Incubation was carried out at 25 ° for 15 rain. Materials B. subtilis ribonuclease was the product reported previouslyll,lL Ribonuclease T1 and T~ were kindly donated b y Dr. H. Okazaki of Central Research Laboratories, Sankyo. G - U - A and G - U - G were the gifts of Dr. T. Ukita, University of Tokyo. Uniformly labeled E14Clvaline (specific activity, 208.5 #C/#mole) was obtained from New England Nuclear. W h a t m a n D E 23 (capacity, I mequiv/g) was obtained from Balston. DEAE-Sephadex A-25 (capacity, 3.5±0.5 mequiv/g; particle size, 4o-12o #) was a product of Pharmacia Fine Chemicals. RESULTS
Inability o] B. subtilis ribonuclease-treated valyl-tRNA to bind to ribosomes As seen in Table I, the ability of B. subtilis ribonuclease-treated valyl-tRNA to bind to ribosomes in the presence of G - U - A and G - U - G was very little when comBiochim. Biophys. Acta. 179 (1969) 97-1o5
ioo
i~. O D A
et al.
TABLE I INABILITY
OF
B. subtilis
RIBONUCLEASE-TREATED
VALYL-tRNA
TO B I N D TO R I B O S O M E S
The reaction m i x t u r e contained 7700 counts/rain of [14C]valyl-tRNA. 6090 c o u n t s / m i n of B. subtilis ribonuclease-treated [i4C]valyl-tRNA, 0.o 5 A u n i t of G - U - A and 0.05 A unit of G - U - G where indicated.
Template
[14C] Valyl-tRNA (counts/min bound to ribosomes)
B. subtilis ribonuclease-treated [14Cjvalyl-tRNA (counts~rain bound to ribosomes)
None G-U-A G-U-G
128 1536 247 °
193 265 297
pared with that of untreated control i14Clvalyl-tRNA. Although not shown in the table, lack of activity of the treated valyl-tRNA in a cell-free protein-synthesizing system directed by poly (Ua,G) was also found, as previously reported with unfractionated t R N A preparations 3.
Restoration of valine acceptor activity of the denatured valine tRNA by renaturation The valine acceptor activity of the ribonuclease-treated valine tRNA was completely inactivated after heating and rapid cooling as shown in Table II. This is in accordance with the previous observation obtained with unfractionated t R N A 3. However, the activity was found to be recovered markedly when the heated t R N A was slowly cooled (20 h) from 85 ° to 3 °0 in a thermos flask. TABLE II RESTORATION
OF VALINE
ACCEPTOR ACTIVITY OF TIlE RIBONUCLEASE-TREATED
VALINE
tRNA
BY
SLOW COOLING
The t R N A (0.o 5 A unit) was dissolved in 0.2 ml of o.2 M NaC1, 0.02 M Tris-HC1 buffer (pH 7.2) and O.Ol M MgCI,, and heated at 85 ° for 5 min. I n the case of rapid cooling, the sample was p u t in an i c e - w a t e r bath.
B. subtilis ribonuclease-treated valine t R N A
Native valine t R N A
[x4ClValine a t t a c h e d (counts/min/o.o 5 ml) Per cent of activity
Control
Heat and rapid cool
Heat and slow cool
Control
Heat and rapid cool
Heat and slow cool
5395 IOO
4946 91. 5
4843 89.7
3287 IOO
179 5.4
216o 65.5
Separation o] valine tRNA ]ragments by DEAE-cellulose column chromatography The ribonuclease-treated valine t R N A was heat-denatured as described in MATERIALS AND METHODS and chromatographed by the use of DEAE-cellulose. The profile of the eluted valine t R N A fragments is shown in Fig. Ia. The two main peaks (II and III) composed of the - C - C - A and the pGp halves of the valine t R N A molecule, respectively, were purified b y rechromatography as shown in Figs. I b and ic. A1Biochim. Biophys. Acta, 179 (1969) 97-1o5
ACCEPTOR ACTIVITY OF VALINE I II I II J
I
Ill
60
80
tRNA II
IV
FRAGMENTS
I0I
I
1.0 t
'4~
1
"
30
40
@
~
20
40
50
70
90
100 110
,
60
80 20 Fraction number
40
60
80
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 valine t R N A f r a g m e n t s , a. S e p a r a t i o n of v a l i n e - t R N A f r a g m e n t s b y D E A E - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y , b. R e c h r o m a t o g r a p h y of F r a g m e n t II b y D E A E - S e p h a d e x A-25 c o l u m n c h r o m a t o g r a p h y , c. R e c h r o m a t o g r a p h y of F r a g m e n t I I I b y D E A E - S e p h a d e x A-25 c o l u m n c h r o m a t o g r a p h y . F o r detail, see MATERIALS AND METHODS.
though not shown in Fig. I, Peaks I and IV were also purified by rechromatography. Peak I consisted of two fragments (Ia and Ib) which were found to be the fragments derived from Component II by further cleavage according to the pattern of the chromatography of ribonuclease T1 digestion products. Peak IV also consisted of two fragments. Fragment IVa, which included two thirds of the total amount of Peak IV, was somewhat similar to Fragment II, and Fragment IVb was found to be the same as Fragment III. These were also suggested by analogy with the products of ribonuclease T 1 digestion. TABLE III RESTORATION OF VALINE ACCEPTOR ACTIVITY BY COMBINING VALINE t R N A FRAGMENTS T h e f r a g m e n t s as specified were dissolved in 0.2 ml of 0.2 M NaC1, 0.02 M Tris-HC1 b u f f e r (pH 7.2) a n d 0.oi M MgCI~. Expt. No.
Addition o[ the lragment Ia A units added/o.2 rot: 0.o3
I
2 3 4 5 6 7 8 9 IO II
Fl*C]Valine accepted (countslmin/o.o5 ml)
lb
II
III
IVa
0.02
o.o97
o.io8
o.o54
+
o
+
6 +
o
+ + + +
Heat and slow cool
+
+ + + N a t i v e valine t R N A , 0.03 A u n i t N a t i v e valine t R N A , 0.06 A u n i t
+ + + +
+
io o 3169 87 1734 3325
No treatment
o
o 2 2 o 128 5 IOO 168 2609 4572
Biockim. Biophys. Acta, 179 (1969) 97 lO5
K. ODA et al.
lO2
Restoration o[ valine acceptor activity ~, combining the [ragments When each fragment was heated and separately cooled slowly (No. i-5) , no valine acceptor activity was restored at all as shown in Table h i . However, surprisingly, when Fragments I I and I I I were mixed and slowly cooled (No. 6), valine acceptor activity was recovered to the extent of approx. 73 % compared with the same A units of native untreated valine tRNA (No. io, Ii). Fragment II could not be replaced by Fragment Ia and Ib, produced by the further cleavage of Fragment I I (No. 7). Furthermore, addition of Fragments Ia and Ib to the mixture of Fragments II and I I I did not alter the extent of the restoration significantly (No. 9). Fragment IVa, which was found to be similar to Fragment n, could be substituted for Fragment I I (No. 8). It should be mentioned that the restoration of activity was very poor when the mixture was assayed without the renaturation procedure.
Temperature dependence o[ the renaturation process ]'he mixture of Fragments II and n I was incubated at 85, 65 and 5°o and slowly cooled to 25 ° in 20 h in each case. The valine acceptor activity was restored to the same extent in every case (7° % as compared with control valine tRNA). In addition it was found that incubation of the mixture at 37 ° for 20 h was enough for maximal restoration of valine acceptor activity. As shown in Fig. 2, the rate of the reactivation was measured by incubating the mixture at different temperatures. The reactivation occurred even at 25 °, but proceeded very slowly. At 37 °, the reactivation proceeded much faster than at 25 ° , and the maximal reactivation was attained after incubation for 2o h. It should be mentioned that when Fragments II and I I I were separately incubated at 37 ° for 2o h, and then mixed followed by innnediate assay, no valine acceptor activity was restored. ! u~
d
i
cl c
6O0
s 4001 o
'~'
500, /i
~ 2 5 °
tt~' /
20oI ;0 '
Time
h
)
i0 '
:0
Concentration of M £ -
lO "1
Fig. 2. Time course of the r e s t o r a t i o n of valine acceptor activity b y reconstitution of F r a g m e n t s I I and I I I . F r a g m e n t s I I and 11I were mixed (o.33 and 0.48 A u n i t / m l respectively) ill 0.2 M NaC1, 0.02 M Tris-HC1 (pH 7.2) and o.o~ M MgC12, and incubated at 37 or 25 °. At the times indicated, aliquots were removed, rapidly cooled and stored at --20 °. Valine acceptor activities were assayed as described previously 4 except t h a t the time of incubation was 5 rain. Fig. 3. R e q u i r e m e n t of Mg ~+ for the r e s t o r a t i o n of valine acceptor activity. F r a g m e n t I I and I I I (0.03 A u n i t each) were mixed in 0.2 ml of 0.2 M NaCI, 0.02 M Tris-HC1 buffer (pH 7,2) and MgClz as specified. I n c u b a t i o n was at 37 ° for 20 h.
Biochim. Biophys. Acta, 179 (1969) 97-1o5
ACCEPTOR ACTIVITY OF VALINE
tRNA
103
FRAGMENTS
Ellect ol magnesium concentration on the restoration o/valine acceptor activity As shown in Fig. 3, the restoration of valine acceptor activity depends on the presence of Mg 2÷. It did not occur at concentrations of Mgz÷ lower than o.ooi M, and the maximal restoration was found at 0.025 M.
E/leer o/ relative concentration o/ Fragments I I and I I I on the restoration o/ valine acceptor activit# As shown in Fig. 4, constant amounts of Fragment I I (or III) were incubated at 37 ° for 20 h with increasing amounts of Fragment I I I (or II), and their acceptor activities were then measured. In both cases, the extent of the restoration of activity increased linearly until the same amount of the complementary fragment was added, and after the addition of a 3-fold excess of the complementary fragment, the increase of activity levelled off. A 14 °/o higher plateau level was attained with Fragment I I I . This m a y indicate that either (i) the length of Fragment I I I is slightly less than that of Fragment II, or (2) Fragment I I I has a greater affinity for Fragment II. a 0"61
. 800 o b
'
~'"
/mr
Native vali
~,f
i
f ~ - ~ ~ i ~ % ~ setPeclted valine t R N A
ff
/mL
"~ 600
o,4--
I
30
40
50
60
70
I
80
90
b
~-~m 4001 ff
2ooy ~
~ ." :t. ~
i
0.2 0.4 0.6 0.8 Amount of complementary fragment (A units/m/.)
~
i
:
~
{I
O.7 ! 30 °
40 °
50 ° 60 o Temperature
70 °
80 °
9() °
Fig. 4. E f f e c t of r e l a t i v e c o n c e n t r a t i o n of F r a g m e n t s I I a n d I I I on t h e r e s t o r a t i o n of v a l i n e acc e p t o r a c t i v i t y . A c o n s t a n t a m o u n t of F r a g m e n t I I or I I I (0.03 A u n i t ) w a s m i x e d w i t h i n c r e a s i n g a m o u n t s of F r a g m e n t I I I or I I r e s p e c t i v e l y in 0.2 ml of 0.2 M NaC1, 0.02 M Tris-HC1 buffer (pH 7.2) a n d o.o i M MgC1 v I n c u b a t i o n w a s a t 37 ° for 20 h. Fig. 5- The t h e r m a l d e n a t u r a t i o n c u r v e of n a t i v e v a l i n e t R N A , B. subtilis r i b o n u c l e a s e - t r e a t e d v a l i n e t R N A a n d i t s { r a g m e n t s in t h e p r e s e n c e of Mg 2+. A b s o r b a n c e a t 26o mt~ w a s m e a s u r e d i n 0.2 M NaC1, 0.02 M T r i s - H C l buffer (pH 7.2) a n d o.oi M MgC1 v The f r a g m e n t s w e re i n c u b a t e d a t 37 ° for 2o h before t h e h e a t t r e a t m e n t was begun. In t h e case of t h e m i x t u r e of F r a g m e n t I I a n d III, t h e s a m p l e c o n t a i n e d 0.33 A u n i t of F r a g m e n t I I a n d 0.36 A u n i t of F r a g m e n t I I I .
Thermal denaturation o / B . subtilis ribonuclease-treated valine tRNA and its/ragments As shown in Fig. 5a, thermal denaturation of the ribonuclease-treated valine tRNA occurred at a lower temperature than that of control native valine tRNA. Biochim. Biophys. Acta, 179 (1969) 97-1o5
lO4
K. ODA et al.
Fig. 5 b shows the thermal denaturation profile of Fragment II, Fragment I I I and their reconstituted molecule. The pattern of the curve of the reconstituted molecule is quite similar to that of B. subtilis ribonuclease-treated valine tRNA, although the percent increase of absorbance of the reconstituted molecule was not as high as that of the ribonuclease-treated valine tRNA. It should be mentioned that Fragment I I or Fragment I I I alone gave distinctive denaturation profiles, and the theoretical curve obtained by taking the mean value of their patterns was not quite the same as that of the reconstituted molecule. In addition, when Fragments I I and I I I were mixed to form the reconstituted molecule, no decrease of absorbance at 260 m,u was detected even after incubation at 37 ° for 20 h. DISCUSSION
The results reported above clearly indicate that valine acceptor activity is completely restored by combining two large fragments (Fragment II, containing the - C - C - A end and Fragment I I I , containing the pGp end) which were obtained b y tLe limited B. subtilis ribonuclease digestion of E. coli valine tRNA. It is essential to combine the two fragments for the reactivation of valine acceptor activity. No acce Ftor activity was restored with either component alone. Furthermore, the reappearance of valine-acceptor activity is temperature and time dependent, and the presence of Mg 2+ is also necessary. Although not shown in RESULTS, it should be mentioned that other divalent cations such as Ca 2+ and Mn 2+ are also active as well as Mg ~+. Furthermore, if the concentration of NaC1 during the renaturation process is increased up to 0.8 M, valine acceptor activity is fully restored even in the absence of the divalent ions (S. NISHIMURA, unpublished data). The association of the two fragments, which is required to accept the amino acid, must take place gradually during the renaturation process. However, no decrease of absorbance at 260 m# was observed during the course of renaturation. This suggests that either (i) interaction of the two fragments that contributes to tile decrease of absorbance occurs in a very small fraction or region as compared with the total molecule, or (2) conformational rearrangements can take place without changing the gross amount of secondary and tertiary structure. At any rate, it should be emphasized that renaturation of this type is a unique reaction since the fragments are composed of relatively short chains of oligonucleotides and the extent of the renaturation is expressed as specific biological activity. The fragments (II and III) are oligonueleotides derived from the - C - C - A and pGp end of the t R N A molecule, respectively. T.be two fragments are approximately the same size, and include most of the sequence of native valine tRNA. This was suggested by comparison of the chromatographic patterns of the ribonuclease T 1 digest of the fragments with that of native valine tRNA. In addition, two-dimensional paper chromatography of a ribonuclease T 2 digest of the fragments 4 showed that Fragment I I contained I mole each of adenosine, N7-methylguanylic acid, N6-methyladenylic acid*, ribothymidylic acid and pseudouridylic acid, and Fragment I I I contained i * W i t h o u t a l k a l i n e t r e a t m e n t , I m o l e of N n - m e t h y l a d e n y l i c a c i d w a s f o u n d i n E. coli v a l i n e t R N A . S i n c e i t is n o t d e r i v e d b y t h e c o n v e r s i o n of N l - m e t h y l a d e n y l i c a c i d o r N S - i s o p e n y e n y l a d e n y l i c a c i d 14,1~, i t is a n e w m i n o r b a s e c o m p o n e n t f o u n d i n t R N A . D e t a i l s of t h i s w i l l b e p u b lished elsewhere.
Biochim. Biophys. Acta, 179 (I969) 9 7 - 1 o 5
ACCEPTOR ACTIVITY OF VALINE
tRNA
FRAGMENTS
Io 5
mole each of guanosine 3',5'-diphosphate and 4-thiouridylic acid. Thus all minor components detected in valise t R N A so far are present in either Fragment II or Fragment III. However, these results do not necessarily mean that two fragments are produced by a single cleavage of valine-tRNA molecule as described by PENSWlCK AND HOLLEY8 and BAYEV et al. 13, although it is most probable case. To clarify this point, a sequential analysis of E. coli valine t R N A is now under investigation. ACKNOWLEDGMENTS
We thank Dr. Sinichiro Esumi, Laboratories of Kaken Chemicals for the largescale isolation of crude E. coli tRNA. The authors are indebted to Dr. G. David Novelli for the critical reading and revision of the manuscript. The work was supported partly by research grant No. 94216, 954025 and 954060 from Ministry of Education. We thank Dr. Dieter $611, Yale University for informing us that better fractionation of E. coli t R N A on benzoylated DEAE-cellulose columns can be achieved at 4 ° .
REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15
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