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Valine transfer ribonucleic acid. I. Chromatographic study of valine tRNA modifications during Bacillus subtilis growth
596
BIOCHIMICA ET BIOPIcfYSICA ACTA
BBA 95723
V A L I N E T R A N S F E R R I B O N U C L E I C ACID I. CHROMATOGRAPHIC STUDY OF V A L I N E t R N A MODIFICATIONS DURING BACILLUS
SUBTILIS
GROWTH
T. HEYMAN, S. SEROR, B. D E S S E A U X AND J, L E G A U L T - D E M A R E
Institut du Radium, Radiobiologie, Bdtiment xIo, 9r-Orsay (France) (Received April 26th, 1967)
SUMMARY
Bacillus subtilis valine tRNA has been found to exist in two molecular forms, one of which is predominant (Form I). For a given amount of total tRNA there is more of this form in exponential-phase than in stationary-phase cells. On the contrary, the concentration of Form I I is the same for both phases. Form n is more rapidly charged than Form I.
INTRODUCTION
The heterogeneity of certain transfer RNA's has been demonstrated by different techniques: chromatography on methylated albumin-kieselguhr 1, countercurrent distribution 2-4, etc. It has also been shown that the relative proportions of the different molecular species of a given tRNA may vary when the physiological state of the cell is altered: e.g. KANo-SuEoKA .aND SUEOKA observed that the chromatographic profile of E. coli leucine t R N A was modified after infection by a T-even phage 5 In Bacillus sublilis, KANEKO AND DOI6 found a change in the ratio of the two peaks of valine t R N A during the early sporulation phase, and LAZZARINI7 published a similar observation for lysine t R N A during late sporulation. In order to determine whether these variations are directly related to the mechanism of sporulation, it appeared interesting to look for analogous modifications in mutants blocked at different stages of sporulation s. In the first part of this work, we have established the proportions of the two valine tRNA's in a wild-type strain at two stages of the growth curve (exponential and stationary, I h) and we have studied the kinetics of the incorporation of valine in each of them. Abbreviations: tRNA, transfer RNA; rRNA, ribosomal RNA.
Biochim. Biophys..dcta, 145 (I967) 596-604
VALINE t R N A MODIFICATIONSIN B. subtilis
597
TECHNIQUES
I. Culture conditions B. subtilis strain W 23 was grown on SCM medium s at 37 ° with vigorous aeration. The cells were harvested either during the exponential phase at an A of 0.500 (at 620 m/~, Bausch and Lomb "Spectronic uo" colorimeter, IO mm diameter tubes), or 60 min after the end of exponential growth, at an A near 1.6; they were washed twice by centrifugation in a o.oi M Tris-HCl buffer (pH 7.2), o.oi M MgCI2, and kept in a frozen state.
2. Preparation o/ t R N A The t R N A was extracted from the cells b y direct phenol treatment according to the method of VoN EHRENSTEIN AND LIPMANN9. However, the W 23 cells appear to be particularly fragile, and a lysis occurs, which renders necessary the following additional steps of purification: (a) three phenol deproteinizations in the presence of 0.5 ~o laurylsulfate; (b) precipitation of ribosomal RNA with i M NaC1; (c) incubation with 2.5 #g/ml of deoxyribonuclease for 30 min at o °, then 5 min at 4 ° in o.oi M Tris-HC1 (pH 7.2), 0.0005 M MgC12, followed by two further phenol treatments. The esterified amino acids were stripped off by incubating the purified RNA for 60 min at 35 ° in a 0.5 M Tris-HC1 buffer, pH 8.8. After each of these steps, the RNA was precipitated with isopropanol in the presence of 2 °/o potassium acetate, pH 5.1. The preparations thus obtained could again be slightly contaminated by rRNA; their t R N A content was determined by chromatography on methylated albumin-kieselguhr according to YAMANE AND SUEOKA 10. RNA was estimated from the absorbance at 260 m~, assuming an extinction coefficient of E 1% 1 c m at 260 m# --~ 240.
3. Preparation o/ the enzyme The aminoacyl synthetase was prepared by the method of YAMANE AND SUEOK~1° from bacteria harvested i h after the end of the exponential growth phase. To the enzyme in 0.02 M phosphate buffer (pH 7-7), 0.5 M NaC1 and 0.006 M mercaptoethanol, glycerol was added to a final concentration of 15 °/o, and the solution was kept at --20 °. The preparation used in the present work contained 600/~g of protein per ml, estimated by means of the FOLIN-LOwR¥ reaction n.
4. Preparation o! valine t R N A The reaction was performed at 37 ° in a o.I M Tris-HC1 buffer solution (pH 7.2), containing o.ooi M mercaptoethanol, o.oi M MgC12 and 0.004 M ATP. The concentrations of enzymes and of radioactively labeled valine are given in RESULTS for each experiment and they are corrected for the amount of tRNA. It was verified that ovaline did not inhibit the reaction. The total incorporation of valine was estimated by precipitating an aliquot of the reaction mixture with trichloroacetic acid at o ° in the presence of 200 #g of serum albumin as carrier. The precipitate was filtered on a Millipore HA membrane, washed with 50 ml of 5 % trichloracetic acid, dried and its radioactivity determined. We used L-[l*C]valine (129 mC/mmole) and DL-[3H~valine (IO C/mmole) both obtained from CEA, Saclay, France. Biochim. Biophys. Acta, 145 (1967) 596-6o4
T. HEYMAN 6[ al.
598
When valine t R N A was prepared for chromatographic analysis, the reaction was stopped at the desired time b y addition of x vol. of phenol. The mixture was stirred for 20 min at room temperature and the RNA was precipitated from the aqueous phase b y addition of isopropanol in the presence of 2 % potassium acetate, p H 5.1.
5. Chromatography on methylated albumin-kieselguhr The columns were prepared with IO g thylated serum albumin. A linear gradient NaC1 and J7 o ml of 0.55 M NaC1 in a 0.05 The t R N A was eluted in about 80 fractions tion was precipitated with trichloroacetic
of Hyflo Super-Cel and 4 ml of 1 % mewas applied between 17o ml of 0.25 M M potassium phosphate buffer, p H 6.5. of 1. 7 ml each. An aliquot of each fracacid and its radioactivity determined.
6. Counting o/radioactivity The samples containing 14C only were counted in a Tracerlab low background gas-flow counter, in which I m#mole of valine yielded 9.6.1o 4 counts/min. The samples containing aH or the two isotopes were counted in a Packard Tri-Carb scintillation counter. ISH]valine alone yielded 242.io 4 counts/min/per m/xmole; when the two isotopes were present, the apparatus was adjusted to reduce the cross-contamination to 6-1o % and the counts were 157-1o 4 countsfmin/per m/zmole for E3H]valine and 17.1o 4 counts/min/per m/zmole for [14C]valine.
RESULTS
Synthesis o/ valine tRNA The incorporation of valine b y the t R N A of exponential or stationary phase cells was measured: (a) in the presence of different enzyme concentrations, after IO min of incubation at 37 ° (Fig. i);
/
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8
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Time ( r a i n )
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Fig. I. S y n t h e s i s of v a l i n e t R N A in t h e p r e s e n c e of d i f f e r e n t c o n c e n t r a t i o n s of e n z y m e . [14C]valine, 4.5 m/zmoles. I n c u b a t i o n t i m e , i o min. I n all figures, t h e a m o u n t s of v a l i n e a n d of e n z y m e are n o r m a l i z e d to IOO # g of t o t a l t R N A . Q - O , e x p o n e n t i a l p h a s e ; × - × , s t a t i o n a r y p h a s e . Fig. 2. S y n t h e s i s of valine t R N A in r e l a t i o n to t i m e of i n c u b a t i o n . [14C]valine, 4.5 m/zmoles. E n z y m e , i8/~g. Q - O , e x p o n e n t i a l p h a s e ; × - × , s t a t i o n a r y p h a s e .
Biochim. Biophys. dcta, 145 (1967) 596-604
VALINE t R N A MODIFICATIONSIN B. subtilis
599
(b) after different periods of incubation, in the presence of excess of enzyme and valine (Fig. 2). The rate of synthesis of valine t R N A was the same, whatever the growth phase in which the cells were harvested. However, in accordance with the results of LAZZARINI v, the m a x i m u m level of incorporation was higher for t R N A from exponentialphase bacteria than for t R N A from stationary-phase bacteria. The influence of magnesium on this reaction has been studied: the incorporation of valine was at a maxim u m for a Mg 2+ concentration of between 0.005 and 0.023 M. It was reduced to about 75 O/joof the m a x i m u m value in the presence of o.I M Mg 2+ and to about 20 °/o in the presence of 0.0023 M Mg 2+.
Chromatography o//ully charged valine tRNA The elution profile of valine t R N A showed two peaks (I and II), as observed b y KANEKO AND Do1 e, Peak I being more pronounced than Peak I I (Figs. 3e and 5d). The higher level of incorporation with RNA from exponential-phase bacteria was due to a net increase of Peak I. On the contrary, the concentration in total t R N A of valine t R N A corresponding to Peak I I was constant in both exponential- and stationary-phase cells (Table I). TABLE I INCORPORATION OF VALINE INTO PEAKS I AND I I UNDER DIFFERENT CONDITIONS The v a l u e s g i v e n are c a l c u l a t e d f r o m t h e a r e a s o c c u p i e d b y e a c h p e a k i n t h e c h r o m a t o g r a m s slaown in t h e f i g u r e s m e n t i o n e d in t h e f i r s t c o l u m n , a n d n o r m a l i z e d for i o o / ~ g of t o t a l t R N A . U p p e r p a r t : s t a t i o n a r y - p h a s e t R N A ; b o t t o m p a r t : e x p o n e n t i a l - p h a s e t R N A . T h e e n z y m e us e d w a s p r e p a r e d f r o m s t a t i o n a r y - p h a s e cells, e x c e p t for *, e n z y m e f r o m e x p o n e n t i a l p h a s e cells.
Fig.
Valine (ml~moles)
Enzyme (l~g)
Incubation time (rain)
Valine incorporated (mtzmoles × lO s) I
II
1/II
3a 3b 3c 3d 4 4 4 4 3e 3e
o.23 o.34 o.77 2. 3 1.55 4.5 4.5 4.5 4.5 4-5
3.6 3.6 3.6 3.6 12.o 12.o 12.o 12.o 12.o 18.o
IO IO IO IO IO I 2.5 6 12 12
o.26 o.38 0.85 1.73 3.5 ° 0.86 1.95 3.4 ° 4.o6 4.Ol
o.31 o.38 o.69 I.IO 1.35 o.67 1.35 1.42 1.52 1.48
o.84 I.OO 1.23 1.57 2.6 1.28 1.44 2. 4 2.67 2.7o
5a 5b 5c 5d 6-1 6-2
0.23 0.45 I.O 4.5 I.O 4.0 4.4
3.6 IO.O 18.o 18.o 18.o
IO IO IO IO 12 IO 12
o.12 0.44 3.2o 6.05 4.35 2.5o 7.0
o.07 0.23 1.32 1.5 ° 1.38 o 1.55
1.71 1.91 2.42 4.04 3.15
15.o*
4.5o
Chromatographic study o/the kinetics o//ormation o/valine tRNA I and I I The elution profiles of partially charged valine t - R N A show that the kinetics Biochim. Biophys. Acta, 145 (1967) 596-6o4
600
T. HEYMAN et al.
of incorporation were not the same for the two species of valine-specific tRNA. Fig. 3 shows the elution profiles of stationary-phase valine t R N A charged in the presence of different concentrations of valine or of enzyme. The ratio of the two peaks varied between 0.84 for a total charge of 0.0057 m#mole of valine (for o.i mg of total tRNA) and 2.6 for a total charge of 0.058 m#mole. 0
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Fig. 3. C h r o m a t o g r a p h y of s t a t i o n a r y - p h a s e valine t R N A at different levels of incorporation. [~H]valine: a, o.23 mffmoles. [14C]valine: b, o.34; c, o.77; d, 2.3; e, 4.5 m f m o l e s . E n z y m e : a, b, c and d: 3.6; e, 1 2 f i g (same result for e w i t h i 8 # g ) .
Fig. 4 shows the levels of incorporation into Peaks I and II in relation to the incubation time. The curves indicate that Form II was fully charged when Form I was charged to only 4 ° O,/o.It was verified that the incorporation rates corresponding Biochim. ]3iophys. Acta, 145 (i967) 5 6 9 - 6 0 4
MODIFICATIONS IN B. subtilis
VALINE t R N A
~338E
6Ol
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, i
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Fig. 4. S y n t h e s i s of s t a t i o n a r y - p h a s e valine t R N A ' s I and n in relation to time of incubation. [14C] valine, 4.5 m # m o l e s . E n z y m e , 12 fig. E a c h point is calculated from t h e areas occupied b y P e a k s I and U after c h r o m a t o g r a p h y of the R N A ' s e x t r a c t e d from the reaction mixtures.
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Fig. 5. C h r o m a t o g r a p h y of e x p o n e n t i a l - p h a s e t R N A at different levels of incorporation. [3H]valine: a, 0.23; b, o.45; c, i . o m/~mole. [14C]valine: d, 4.5 m # m o l e s . E n z y m e : a, 3.6; b, io.o; c, and d, I 8 ~Ag.
Biochim. Biophys. Acla, 145 (1967) 5 9 6 6 o 4
602
T. HEYMANet al.
to both peaks were not modified when the concentration of ATP in the reaction medium was increased from 0.004 to 0.008 M13. In the case of exponential-phase tRNA, the first peak was always more pronounced, even when tile incorporation was 1/4o of the maximum. However, as for exponential-phase tRNA, the I/II ratio increased when the total incorporation was increased, which indicates a faster synthesis of valyl t R N A II (Fig. 5). If the tRNA was charged with IaHlvaline at a limiting concentration, and then with I14C]valine at a concentration which allowed maximum incorporation, the distribution of the radioactivity in the elution profile clearly showed that Form II was fully charged before Form I (Fig. 6). The same results were obtained with the enzyme from either exponential- or stationary-phase cells.
A260 mN
3H _c
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0
10
20
30
40 50 Fraction No
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70
Fig. 6. Chromatography of exponential-phase valine tRNA charged in two steps: (I) ?H]valine, i m#mole. (2) [14C]valine, 4 m/~moles.
DISCUSSION
The experiments reported here show that B. sublilis valine t R N A exists in two molecular forms, one of which is predominant (Form I). For a given amount of total tRNA, there is more of this Form I in exponential-phase bacteria than in stationary-phase bacteria. On the contrary, the concentration of Form I I is the same for both phases. In addition, Form II is more rapidly charged than Form I. These results appear to be different from the findings of KANRKO .aND DOle: these authors observed the predominance of Form I in exponential-phase tRNA and of Form II in tRNA from cells harvested after i h in the stationary phase (early sporulation); the chromatographic profile returned to the exponential-phase type after 4 h in stationary phase (late sporulation). This discrepancy may be explained by the fact that in this work the tRNA from the stationary phase was only partially charged. Biochim. Biophys. Acta, i45 (I967) 596-604
VALINE t R N A
6o 3
MODIFICATIONS IN B . subtilis
In fact, K A N E K O AND DoI used the same amount of valine, expressed in terms of radioactivity (2.5/zC) to charge o.5-1.o #g of total tRNA. In the case of stationaryphase tRNA, t h e y employed EaHlvaline, 1.2 C/mmole, i.e. 0.2-0. 4 m/~moles of valine for IOO #g of tRNA. In similar conditions (our Figs. 3a and 3b) we found profiles very close to the results of KANEKO AND DOI; we also demonstrated that in this case Peak I was grossly under-estimated, as the same stationary-phase t R N A exhibited the profile shown in our Fig. 3e when fully charged with the adequate amount of valine. On the other hand, the counts/min values given in Fig. 3 b y KANEKO AND DoI show that the m a x i m u m of Peak ]I occurred at more than IOOOcounts/min for "early" and at about 15o counts/min for "late" sporulation tRNA; as the A curve is almost the same in both cases one must conclude that there was six times less "active" t R N A Val in the "late" sample. As apparently the amount of input [aH~valine was not modified accordingly, the ratio of valine to RNA was multiplied b y the same factor and amounted thus to 1.2 m/zmoles for IOO #g of tRNA, precisely the same value which was used for E14CJvaline and exponential phase tRNA. Under these conditions, it is not surprising that the aH profile "returned" to the log-phase type in late sporulation phase. Many questions remain to be answered, for example it is not yet known whether there is only one valyl synthetase or two enzymes corresponding to each of the two valine-specific tRNA's, as in the case of aspartyl- and of phenylalanyl-tRNA's*. It should be interesting also to study the possible differences between the two valine t R N A ' s with respect to their specificity of incorporation with heterologous enzymes 5 or with regard to transfer specificity in sub-cellular systems. Another aspect of this problem lies in the difference observed on the amounts of Peak I between exponential-phase and stationary-phase bacteria. This difference could be due either to an actual decrease of the concentration of Form I in stationaryphase tRNA, or to some kind of partial inactivation of this form e.g. through CCA elimination (J. P. EBEL personal communication). As the technique used here allowed us to measure only the amount of functional valine-specific tRNA, it is not yet possible to discriminate between these two possibilities, nor to relate them to a regulation mechanism. These problems are currently under study.
ACKNOWLEDGEMENTS
We are indebted to Dr. R. H. DoI for the gift of B. subtilis W23. We are grateful to Drs. R. H. DoI and J. P. WALLER, and to Professor P. SCHAEFFER for m a n y helpful discussions.
REFERENCES i 2 3 4 5
•. SUEOKA AND T. YAMANE, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1454. J. APGAR, R. W. HOLLEY AND S. H. MERRIL, Biochim. Biophys. Acta, 53 (1961) 220. R. THIEBE AND H. G. ZACHAU, Biochim. Biophys. Acta, lO 3 (1965) 568. W. E. BARNETT AND J. L. EPLER, Proc. Natl. Acad. Sci. U. S., 55 (1966) 184. T. KANO-SUEOKA AND ~N~'. SUEOKA, J. Mol. Biol., 20 (1966) 183.
Biochim. Biophys. Acta, 145 (1967) 596-604
6o4
T. I-IEYMAN et
al.
I. t{ANEKO AND ]~. H. DoI, Proc. Natl. Acad. Sci. U.S., 55 (1966) 564 . R. A. LAZZARINI, Proc. Natl. Acad. Sci. U.S., 56 (1966) 185. A. RYTER, H. ION~SCO AND P. SCHAEFFER, C.R. Acad. Sci., 252(1961) 3675 . G. VON EHRENSTEIN AND V. LIPMANN, Proc. Natl. Acad. U.S., 47 (1961) 941. T. YAMANE AND ~]'. SUEOKA, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) lO93. O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 12 P. HELE, Biochem. J . , 81 (1961) 339. 13 N. J. GARTLAND AND ~N]'.SUEOKA, Proc. Natl. Acad. Sci. U.S., 55 (1966) 948.
6 7 8 9 io ii
Biochim. Biophys. Acta, 145 (1967) 596-604
BIOCHIMICA ET BIOPIcfYSICA ACTA
BBA 95723
V A L I N E T R A N S F E R R I B O N U C L E I C ACID I. CHROMATOGRAPHIC STUDY OF V A L I N E t R N A MODIFICATIONS DURING BACILLUS
SUBTILIS
GROWTH
T. HEYMAN, S. SEROR, B. D E S S E A U X AND J, L E G A U L T - D E M A R E
Institut du Radium, Radiobiologie, Bdtiment xIo, 9r-Orsay (France) (Received April 26th, 1967)
SUMMARY
Bacillus subtilis valine tRNA has been found to exist in two molecular forms, one of which is predominant (Form I). For a given amount of total tRNA there is more of this form in exponential-phase than in stationary-phase cells. On the contrary, the concentration of Form I I is the same for both phases. Form n is more rapidly charged than Form I.
INTRODUCTION
The heterogeneity of certain transfer RNA's has been demonstrated by different techniques: chromatography on methylated albumin-kieselguhr 1, countercurrent distribution 2-4, etc. It has also been shown that the relative proportions of the different molecular species of a given tRNA may vary when the physiological state of the cell is altered: e.g. KANo-SuEoKA .aND SUEOKA observed that the chromatographic profile of E. coli leucine t R N A was modified after infection by a T-even phage 5 In Bacillus sublilis, KANEKO AND DOI6 found a change in the ratio of the two peaks of valine t R N A during the early sporulation phase, and LAZZARINI7 published a similar observation for lysine t R N A during late sporulation. In order to determine whether these variations are directly related to the mechanism of sporulation, it appeared interesting to look for analogous modifications in mutants blocked at different stages of sporulation s. In the first part of this work, we have established the proportions of the two valine tRNA's in a wild-type strain at two stages of the growth curve (exponential and stationary, I h) and we have studied the kinetics of the incorporation of valine in each of them. Abbreviations: tRNA, transfer RNA; rRNA, ribosomal RNA.
Biochim. Biophys..dcta, 145 (I967) 596-604
VALINE t R N A MODIFICATIONSIN B. subtilis
597
TECHNIQUES
I. Culture conditions B. subtilis strain W 23 was grown on SCM medium s at 37 ° with vigorous aeration. The cells were harvested either during the exponential phase at an A of 0.500 (at 620 m/~, Bausch and Lomb "Spectronic uo" colorimeter, IO mm diameter tubes), or 60 min after the end of exponential growth, at an A near 1.6; they were washed twice by centrifugation in a o.oi M Tris-HCl buffer (pH 7.2), o.oi M MgCI2, and kept in a frozen state.
2. Preparation o/ t R N A The t R N A was extracted from the cells b y direct phenol treatment according to the method of VoN EHRENSTEIN AND LIPMANN9. However, the W 23 cells appear to be particularly fragile, and a lysis occurs, which renders necessary the following additional steps of purification: (a) three phenol deproteinizations in the presence of 0.5 ~o laurylsulfate; (b) precipitation of ribosomal RNA with i M NaC1; (c) incubation with 2.5 #g/ml of deoxyribonuclease for 30 min at o °, then 5 min at 4 ° in o.oi M Tris-HC1 (pH 7.2), 0.0005 M MgC12, followed by two further phenol treatments. The esterified amino acids were stripped off by incubating the purified RNA for 60 min at 35 ° in a 0.5 M Tris-HC1 buffer, pH 8.8. After each of these steps, the RNA was precipitated with isopropanol in the presence of 2 °/o potassium acetate, pH 5.1. The preparations thus obtained could again be slightly contaminated by rRNA; their t R N A content was determined by chromatography on methylated albumin-kieselguhr according to YAMANE AND SUEOKA 10. RNA was estimated from the absorbance at 260 m~, assuming an extinction coefficient of E 1% 1 c m at 260 m# --~ 240.
3. Preparation o/ the enzyme The aminoacyl synthetase was prepared by the method of YAMANE AND SUEOK~1° from bacteria harvested i h after the end of the exponential growth phase. To the enzyme in 0.02 M phosphate buffer (pH 7-7), 0.5 M NaC1 and 0.006 M mercaptoethanol, glycerol was added to a final concentration of 15 °/o, and the solution was kept at --20 °. The preparation used in the present work contained 600/~g of protein per ml, estimated by means of the FOLIN-LOwR¥ reaction n.
4. Preparation o! valine t R N A The reaction was performed at 37 ° in a o.I M Tris-HC1 buffer solution (pH 7.2), containing o.ooi M mercaptoethanol, o.oi M MgC12 and 0.004 M ATP. The concentrations of enzymes and of radioactively labeled valine are given in RESULTS for each experiment and they are corrected for the amount of tRNA. It was verified that ovaline did not inhibit the reaction. The total incorporation of valine was estimated by precipitating an aliquot of the reaction mixture with trichloroacetic acid at o ° in the presence of 200 #g of serum albumin as carrier. The precipitate was filtered on a Millipore HA membrane, washed with 50 ml of 5 % trichloracetic acid, dried and its radioactivity determined. We used L-[l*C]valine (129 mC/mmole) and DL-[3H~valine (IO C/mmole) both obtained from CEA, Saclay, France. Biochim. Biophys. Acta, 145 (1967) 596-6o4
T. HEYMAN 6[ al.
598
When valine t R N A was prepared for chromatographic analysis, the reaction was stopped at the desired time b y addition of x vol. of phenol. The mixture was stirred for 20 min at room temperature and the RNA was precipitated from the aqueous phase b y addition of isopropanol in the presence of 2 % potassium acetate, p H 5.1.
5. Chromatography on methylated albumin-kieselguhr The columns were prepared with IO g thylated serum albumin. A linear gradient NaC1 and J7 o ml of 0.55 M NaC1 in a 0.05 The t R N A was eluted in about 80 fractions tion was precipitated with trichloroacetic
of Hyflo Super-Cel and 4 ml of 1 % mewas applied between 17o ml of 0.25 M M potassium phosphate buffer, p H 6.5. of 1. 7 ml each. An aliquot of each fracacid and its radioactivity determined.
6. Counting o/radioactivity The samples containing 14C only were counted in a Tracerlab low background gas-flow counter, in which I m#mole of valine yielded 9.6.1o 4 counts/min. The samples containing aH or the two isotopes were counted in a Packard Tri-Carb scintillation counter. ISH]valine alone yielded 242.io 4 counts/min/per m/xmole; when the two isotopes were present, the apparatus was adjusted to reduce the cross-contamination to 6-1o % and the counts were 157-1o 4 countsfmin/per m/zmole for E3H]valine and 17.1o 4 counts/min/per m/zmole for [14C]valine.
RESULTS
Synthesis o/ valine tRNA The incorporation of valine b y the t R N A of exponential or stationary phase cells was measured: (a) in the presence of different enzyme concentrations, after IO min of incubation at 37 ° (Fig. i);
/
@
E
~0.100-
E
E
0100-
/J, -
8
%
1I0 Enzyme
/
E
,
2~ (,/LQ)
0 0
° f
+ ~ - -
5
Time ( r a i n )
10
Fig. I. S y n t h e s i s of v a l i n e t R N A in t h e p r e s e n c e of d i f f e r e n t c o n c e n t r a t i o n s of e n z y m e . [14C]valine, 4.5 m/zmoles. I n c u b a t i o n t i m e , i o min. I n all figures, t h e a m o u n t s of v a l i n e a n d of e n z y m e are n o r m a l i z e d to IOO # g of t o t a l t R N A . Q - O , e x p o n e n t i a l p h a s e ; × - × , s t a t i o n a r y p h a s e . Fig. 2. S y n t h e s i s of valine t R N A in r e l a t i o n to t i m e of i n c u b a t i o n . [14C]valine, 4.5 m/zmoles. E n z y m e , i8/~g. Q - O , e x p o n e n t i a l p h a s e ; × - × , s t a t i o n a r y p h a s e .
Biochim. Biophys. dcta, 145 (1967) 596-604
VALINE t R N A MODIFICATIONSIN B. subtilis
599
(b) after different periods of incubation, in the presence of excess of enzyme and valine (Fig. 2). The rate of synthesis of valine t R N A was the same, whatever the growth phase in which the cells were harvested. However, in accordance with the results of LAZZARINI v, the m a x i m u m level of incorporation was higher for t R N A from exponentialphase bacteria than for t R N A from stationary-phase bacteria. The influence of magnesium on this reaction has been studied: the incorporation of valine was at a maxim u m for a Mg 2+ concentration of between 0.005 and 0.023 M. It was reduced to about 75 O/joof the m a x i m u m value in the presence of o.I M Mg 2+ and to about 20 °/o in the presence of 0.0023 M Mg 2+.
Chromatography o//ully charged valine tRNA The elution profile of valine t R N A showed two peaks (I and II), as observed b y KANEKO AND Do1 e, Peak I being more pronounced than Peak I I (Figs. 3e and 5d). The higher level of incorporation with RNA from exponential-phase bacteria was due to a net increase of Peak I. On the contrary, the concentration in total t R N A of valine t R N A corresponding to Peak I I was constant in both exponential- and stationary-phase cells (Table I). TABLE I INCORPORATION OF VALINE INTO PEAKS I AND I I UNDER DIFFERENT CONDITIONS The v a l u e s g i v e n are c a l c u l a t e d f r o m t h e a r e a s o c c u p i e d b y e a c h p e a k i n t h e c h r o m a t o g r a m s slaown in t h e f i g u r e s m e n t i o n e d in t h e f i r s t c o l u m n , a n d n o r m a l i z e d for i o o / ~ g of t o t a l t R N A . U p p e r p a r t : s t a t i o n a r y - p h a s e t R N A ; b o t t o m p a r t : e x p o n e n t i a l - p h a s e t R N A . T h e e n z y m e us e d w a s p r e p a r e d f r o m s t a t i o n a r y - p h a s e cells, e x c e p t for *, e n z y m e f r o m e x p o n e n t i a l p h a s e cells.
Fig.
Valine (ml~moles)
Enzyme (l~g)
Incubation time (rain)
Valine incorporated (mtzmoles × lO s) I
II
1/II
3a 3b 3c 3d 4 4 4 4 3e 3e
o.23 o.34 o.77 2. 3 1.55 4.5 4.5 4.5 4.5 4-5
3.6 3.6 3.6 3.6 12.o 12.o 12.o 12.o 12.o 18.o
IO IO IO IO IO I 2.5 6 12 12
o.26 o.38 0.85 1.73 3.5 ° 0.86 1.95 3.4 ° 4.o6 4.Ol
o.31 o.38 o.69 I.IO 1.35 o.67 1.35 1.42 1.52 1.48
o.84 I.OO 1.23 1.57 2.6 1.28 1.44 2. 4 2.67 2.7o
5a 5b 5c 5d 6-1 6-2
0.23 0.45 I.O 4.5 I.O 4.0 4.4
3.6 IO.O 18.o 18.o 18.o
IO IO IO IO 12 IO 12
o.12 0.44 3.2o 6.05 4.35 2.5o 7.0
o.07 0.23 1.32 1.5 ° 1.38 o 1.55
1.71 1.91 2.42 4.04 3.15
15.o*
4.5o
Chromatographic study o/the kinetics o//ormation o/valine tRNA I and I I The elution profiles of partially charged valine t - R N A show that the kinetics Biochim. Biophys. Acta, 145 (1967) 596-6o4
600
T. HEYMAN et al.
of incorporation were not the same for the two species of valine-specific tRNA. Fig. 3 shows the elution profiles of stationary-phase valine t R N A charged in the presence of different concentrations of valine or of enzyme. The ratio of the two peaks varied between 0.84 for a total charge of 0.0057 m#mole of valine (for o.i mg of total tRNA) and 2.6 for a total charge of 0.058 m#mole. 0
0
a
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E
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u
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01
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~.
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0.1
10
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30
40
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Fraction NO
60
70
!2 ?
~" T
20
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& cl
o
c_
o~
"~ 0,2-
4~ff. ff ~
o
I c× o~ 1
0
10
20
30
40
50
Fraction No
60
70
Fig. 3. C h r o m a t o g r a p h y of s t a t i o n a r y - p h a s e valine t R N A at different levels of incorporation. [~H]valine: a, o.23 mffmoles. [14C]valine: b, o.34; c, o.77; d, 2.3; e, 4.5 m f m o l e s . E n z y m e : a, b, c and d: 3.6; e, 1 2 f i g (same result for e w i t h i 8 # g ) .
Fig. 4 shows the levels of incorporation into Peaks I and II in relation to the incubation time. The curves indicate that Form II was fully charged when Form I was charged to only 4 ° O,/o.It was verified that the incorporation rates corresponding Biochim. ]3iophys. Acta, 145 (i967) 5 6 9 - 6 0 4
MODIFICATIONS IN B. subtilis
VALINE t R N A
~338E
6Ol
/
, i
~ou
c,
lj
fo Time (min)
Fig. 4. S y n t h e s i s of s t a t i o n a r y - p h a s e valine t R N A ' s I and n in relation to time of incubation. [14C] valine, 4.5 m # m o l e s . E n z y m e , 12 fig. E a c h point is calculated from t h e areas occupied b y P e a k s I and U after c h r o m a t o g r a p h y of the R N A ' s e x t r a c t e d from the reaction mixtures.
3 -3
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i
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70
-30
'.t.~
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03
..
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-20
~
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-10
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o
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.
'%%...~
*.%%.
d..o
r"" , ~0 20 3b 20 ~0 6b " ~ 70 Fraction
No.
0
0
0
1'0 2'0 3'0 4'0 ~0 6'0 7b Fraction
No.
Fig. 5. C h r o m a t o g r a p h y of e x p o n e n t i a l - p h a s e t R N A at different levels of incorporation. [3H]valine: a, 0.23; b, o.45; c, i . o m/~mole. [14C]valine: d, 4.5 m # m o l e s . E n z y m e : a, 3.6; b, io.o; c, and d, I 8 ~Ag.
Biochim. Biophys. Acla, 145 (1967) 5 9 6 6 o 4
602
T. HEYMANet al.
to both peaks were not modified when the concentration of ATP in the reaction medium was increased from 0.004 to 0.008 M13. In the case of exponential-phase tRNA, the first peak was always more pronounced, even when tile incorporation was 1/4o of the maximum. However, as for exponential-phase tRNA, the I/II ratio increased when the total incorporation was increased, which indicates a faster synthesis of valyl t R N A II (Fig. 5). If the tRNA was charged with IaHlvaline at a limiting concentration, and then with I14C]valine at a concentration which allowed maximum incorporation, the distribution of the radioactivity in the elution profile clearly showed that Form II was fully charged before Form I (Fig. 6). The same results were obtained with the enzyme from either exponential- or stationary-phase cells.
A260 mN
3H _c
°Q2
.<
u
0.1
05
0
10
20
30
40 50 Fraction No
60
5
70
Fig. 6. Chromatography of exponential-phase valine tRNA charged in two steps: (I) ?H]valine, i m#mole. (2) [14C]valine, 4 m/~moles.
DISCUSSION
The experiments reported here show that B. sublilis valine t R N A exists in two molecular forms, one of which is predominant (Form I). For a given amount of total tRNA, there is more of this Form I in exponential-phase bacteria than in stationary-phase bacteria. On the contrary, the concentration of Form I I is the same for both phases. In addition, Form II is more rapidly charged than Form I. These results appear to be different from the findings of KANRKO .aND DOle: these authors observed the predominance of Form I in exponential-phase tRNA and of Form II in tRNA from cells harvested after i h in the stationary phase (early sporulation); the chromatographic profile returned to the exponential-phase type after 4 h in stationary phase (late sporulation). This discrepancy may be explained by the fact that in this work the tRNA from the stationary phase was only partially charged. Biochim. Biophys. Acta, i45 (I967) 596-604
VALINE t R N A
6o 3
MODIFICATIONS IN B . subtilis
In fact, K A N E K O AND DoI used the same amount of valine, expressed in terms of radioactivity (2.5/zC) to charge o.5-1.o #g of total tRNA. In the case of stationaryphase tRNA, t h e y employed EaHlvaline, 1.2 C/mmole, i.e. 0.2-0. 4 m/~moles of valine for IOO #g of tRNA. In similar conditions (our Figs. 3a and 3b) we found profiles very close to the results of KANEKO AND DOI; we also demonstrated that in this case Peak I was grossly under-estimated, as the same stationary-phase t R N A exhibited the profile shown in our Fig. 3e when fully charged with the adequate amount of valine. On the other hand, the counts/min values given in Fig. 3 b y KANEKO AND DoI show that the m a x i m u m of Peak ]I occurred at more than IOOOcounts/min for "early" and at about 15o counts/min for "late" sporulation tRNA; as the A curve is almost the same in both cases one must conclude that there was six times less "active" t R N A Val in the "late" sample. As apparently the amount of input [aH~valine was not modified accordingly, the ratio of valine to RNA was multiplied b y the same factor and amounted thus to 1.2 m/zmoles for IOO #g of tRNA, precisely the same value which was used for E14CJvaline and exponential phase tRNA. Under these conditions, it is not surprising that the aH profile "returned" to the log-phase type in late sporulation phase. Many questions remain to be answered, for example it is not yet known whether there is only one valyl synthetase or two enzymes corresponding to each of the two valine-specific tRNA's, as in the case of aspartyl- and of phenylalanyl-tRNA's*. It should be interesting also to study the possible differences between the two valine t R N A ' s with respect to their specificity of incorporation with heterologous enzymes 5 or with regard to transfer specificity in sub-cellular systems. Another aspect of this problem lies in the difference observed on the amounts of Peak I between exponential-phase and stationary-phase bacteria. This difference could be due either to an actual decrease of the concentration of Form I in stationaryphase tRNA, or to some kind of partial inactivation of this form e.g. through CCA elimination (J. P. EBEL personal communication). As the technique used here allowed us to measure only the amount of functional valine-specific tRNA, it is not yet possible to discriminate between these two possibilities, nor to relate them to a regulation mechanism. These problems are currently under study.
ACKNOWLEDGEMENTS
We are indebted to Dr. R. H. DoI for the gift of B. subtilis W23. We are grateful to Drs. R. H. DoI and J. P. WALLER, and to Professor P. SCHAEFFER for m a n y helpful discussions.
REFERENCES i 2 3 4 5
•. SUEOKA AND T. YAMANE, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1454. J. APGAR, R. W. HOLLEY AND S. H. MERRIL, Biochim. Biophys. Acta, 53 (1961) 220. R. THIEBE AND H. G. ZACHAU, Biochim. Biophys. Acta, lO 3 (1965) 568. W. E. BARNETT AND J. L. EPLER, Proc. Natl. Acad. Sci. U. S., 55 (1966) 184. T. KANO-SUEOKA AND ~N~'. SUEOKA, J. Mol. Biol., 20 (1966) 183.
Biochim. Biophys. Acta, 145 (1967) 596-604
6o4
T. I-IEYMAN et
al.
I. t{ANEKO AND ]~. H. DoI, Proc. Natl. Acad. Sci. U.S., 55 (1966) 564 . R. A. LAZZARINI, Proc. Natl. Acad. Sci. U.S., 56 (1966) 185. A. RYTER, H. ION~SCO AND P. SCHAEFFER, C.R. Acad. Sci., 252(1961) 3675 . G. VON EHRENSTEIN AND V. LIPMANN, Proc. Natl. Acad. U.S., 47 (1961) 941. T. YAMANE AND ~]'. SUEOKA, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) lO93. O. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 12 P. HELE, Biochem. J . , 81 (1961) 339. 13 N. J. GARTLAND AND ~N]'.SUEOKA, Proc. Natl. Acad. Sci. U.S., 55 (1966) 948.
6 7 8 9 io ii
Biochim. Biophys. Acta, 145 (1967) 596-604