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Role of valine transfer RNA in control of RNA synthesis in Escherichia coli
PRELIMINARY NOTES
515
BBA 91247
Role of valine transfer R N A in control of RNA synthesis in Escherichia coli It is well established that amino acids are involved in the regulation of RNA synthesis in bacteria 1. In addition, it has been shown that in the case of at least 2 amino acids, their participation in RNA control was dependent on a functional aminoacyl-tRNA synthetase *,a. However, it was not apparent from these studies whether the formation of the amino acid-adenylate enzyme complex or of aminoacyl-tRNA was the necessary step for regulation of RNA synthesis. We now report evidence for the participation of valine tRNA in the control of RNA synthesis in Escherichia coli W strain M42-II, an isoleucine-valine auxotroph stringent for RNA control. For these experiments, DL-x-aminobutyrate and DL-threo~-amino-fl-chlorobutyrate (chlorobutyrate) were used. These valine analogues are activated by valyl-tRNA synthetase 4 (L-valine :tRNA ligase (AMP), EC 6.1.1.9) but only chlorobutyrate is transferred to valine tRNA 5. The cells were grown at 37 ° with aeration in minimal medium e supplemented with L-isoleucine (40 #g/ml), L-valine (80/~g/ml), L-phenylalanine (20/zg/ml), uracil (20 #g/ml), sodium acetate (0.2 %) and glucose (0.2 %). The cells were harvested at a density equivalent to 15o K l e t t Summerson colorimeter units, washed twice with minimal medium, and resuspended in minimal medium supplemented with L-isoleucine (4° #g/ml). The cells were placed in flasks at a density equal to 40 Klett-Summerson colorimeter units. L-Valine (4° #g/ml) was added to one set of flasks, a second set received no valine, and chlorobutyrate (60 #g/ml) and aminobutyrate (30o/~g/ml) were added to a third and fourth set of flasks, respectively. RNA and protein synthesis was measured by the uptake of E14Clphenylalanine and [14C]uracil into trichloroacetic acid-insoluble material 5. As expected, RNA and protein synthesis were completely blocked when the auxotroph was deprived of valine (Fig. I). However, the addition of chlorobutyrate to the valine-starved culture resulted in considerable synthesis of RNA while protein synthesis was essentially blocked. The analogue caused approximately a 5-fold uncoupling of RNA and protein synthesis. In contrast, no RNA or protein synthesis TABLE
I
CHEMICAL DETERMINATION OF R N A AND PROTEIN SYNTHESIZED UPON ADDITION OF VALINE AND CHLOROBUTYRATE IN E . coli STRAIN M 4 2 - I I The cells were grown and harvested as described in the text. They were resuspended in minimal m e d i u m w i t h L - i s o l e u c i n e (4 ° / , g / m l ) a n d L - v a l i n e (4 ° / z g / m l ) o r c h l o r o b u t y r a t e ( 6 0 / z g / m l ) . S a m ples were removed and chemical determinations were made for protein s and RNA*. The values for RNA and protein were normalized to the values obtained at zero time.
Time after addition (min)
Compound added
RNA
o 3° 60 o 3o 60
Valine Valine Valine Chlorobutyrate Chlorobutyrate Chlorobutyrate
i.oo 1.3o 2.5 ° I.oo I.I4 1.71
Protein
RNA Protein
i.oo 1.34 2. I8 I.OO I.OO i.oo
i.oo 0.97 I.iO I.OO I.I4 1.71
BiocMm. Biophys. Acta, 179 ( 1 9 6 9 ) 5 1 5 - 5 1 7
516
PRELIMINARY NOTES
I000
I
I
I
I
~ooo
I
I / 6
/
900 --
F
c E ~-- 7 0 0
o
•
z
-
~
w
500
-
~
X~
Q: a:
o
300
300
lO(
o
15
30 INCUBATION
45 TIME (MIN)
60
o
J
I
15 30 INCUBATION TIME
f
45
60
(giN)
Fig. I. Effect of ~-aminobutyrate and chlorobutyrate on RNA and protein synthesis. The cells were harvested during exponential growth and resuspended in minimal medium with supplementation as described in the text. RNA and protein synthesis was followed by the uptake of [14CJuracil (15 #g/ml) and DL-[14C]phenylalanine (3°/zg/ml). To each flask containing [t4CJuracil was added DL-[x2C~phenylalanine (3o/zg/ml) and the flasks with E14C~phenylalanine received [z2Cluracil (15/~g/ml). Samples were removed and the radioactivity incorporated into trichloroacetic acid-insoluble material measured as described previouslys. Valine excess: C), RNA; 0 , protein. No valine: &, RNA; A, l~rotein. No valine plus ct-aminobutyrate: ~ , RNA; II, protein. No valine plus chlorobutyrate: ×, RNA; ®, protein. Fig. 2. Effect of valine starvation and chlorobutyrate on lipid synthesis. The cells were harvested during exponential growth and resuspended in minimal medium plus ~]4C]acetate (o.2 %) and other supplements as described in the text. Lipid synthesis was measured by the incorporation of [x4C~acetate as determined by the method of SOKA'vVAet aLL 0 , valine (4o/tg/m]); A, no valine or no valine plus ~-aminobutyrate (3oo/*g/ml); ×, no valine plus chlorobutyrate (60 btg/ml).
occurred when aminobutyrate was added to the cultures starved of valine. To test the validity of these isotope incorporation studies, chemical determinations of RNA and protein were performed on samples from cultures grown as described in the previous experiment. As shown in Table I, the addition of chlorobutyrate to a culture deprived of valine resulted in a 7 ° % increase in the RNA to protein ratio. It has recently been suggested that the synthesis of lipids in E. coli is regulated by amino acids in a similar manner to RNAL To test this hypothesis, lipid synthesis was followed in strain M42-II grown in the absence of valine, with and without chlorobutyrate. Lipid synthesis was measured by the incorporation of E14C~acetate as described previously 7 except that the radioactivity of the samples was determined with a Packard scintillation counter. The data in Fig. 2 show that [14C~acetate incorporation was essentially blocked during valine starvation. Significantly, the addition of chlorobutyrate allowed for appreciable lipid formation in the absence of valine. Biochim. Biophys. Acta, 179 (1969) 515-517
517
PRELIMINARY NOTES
The results reported here show that although x-aminobutyrate and chlorobutyrate are activated by valyl-tRNA synthetase, only the analogue which can attach to valine tRNA can support RNA synthesis without the concomitant synthesis of protein. These observations confirm the earlier report a that valine must be activated in order to participate in RNA control and they further suggest that valine must be transferred to tRNA before it can support RNA synthesis. The data further indicate that tRNA may also be involved in the postulated regulation of lipid biosynthesis by amino acidsL We thank M. Rabinovitz for chlorobutyrate. L. S. W. is a Postdoctoral fellow of the American Cancer Society. This work was supported by a grant from the Joint Awards Council of the State University of New York.
Biochemistry Section, Department o/ Biological Sciences, State University of New York, Stony Brook, N. Y. (U.S.A.)
L . S . WILLIAMS M. FREUNDLICH
z G. EDLIN AND P. BRODA, Bacteriol. Rev., 32 (1968) 206. 2 W. L. FANGMAN AND F. C. •EIDHARDT, J. Biol. Chem., 239 (1964) 1844. 3 L. EIDLIC AND F. C. NEIDIIARDT, J. Bacteriol., 89 (1965) 706. 4 F. B. BERGMANN, P. BERG AND M. DIECKMANN, J. Biol. Chem., 236 (1961) 1735. 5 M. FREUNDLICrI, Science, 157 (1967) 823. 6 B. D. DAVIS AND E. S. MII~GIOLI, J. Bacteriol., 6o (195o) 17. 7 Y. SOKAWA, E. NAKAO AND Y. KAZlRO, Biochem. Biophys. Res. Commun., 33 (1968) lO8. 8 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 9 A. MARSHAK AND H. J. VOGEL, J. Biol. Chem., I89 (1951) 597.
Received January I7th, I969 Biochim. Biophys, Acta, I79 (I969) 515-517
BBA 91244 The presence of 2-methylthio-N6.(A2.isopentenyl)adenosine phenylalanine tronsfer RNA's from Escherichia coli
in serine ond
We previously reported the characterization of a new modified nucleoside in Escherichia coli tRNA Tyr as 2-methylthio-N6-isopentenyladenosine1. It is of particular interest that this nucleoside is located next to the 3'-end of the anticodon of E. coli tRNA Tyr (ref, 2). This nucleoside was also independently isolated and identified as one of the cytokinin active components from the total mixture of E. coli tRNA by BURROWS et al. 3. The presence of N6-isopentenyladenosine in exactly the same position (next to the anticodon) of yeast tRNA Tyr (ref. 4) and tRNA set (ref. 5) raised the question of whether or not E. coli tRNA set as well as other species of tRNA from E. coli contains 2-methylthio-Ne-isopentenyladenosine. This paper briefly reports the identification of 2-methylthio-N*-isopentenyladenosine in tRNA s~r and tRNA Ph~ from E. coll. E. coli tRNA Ph~ was prepared as described previously s. E. coli tRNA se~ was first fractionated into three species (tRNA s~ and tRNAS~ r for codons of UC series, Biochim, Biophys. Acta, 179 (1969) 517-52o
515
BBA 91247
Role of valine transfer R N A in control of RNA synthesis in Escherichia coli It is well established that amino acids are involved in the regulation of RNA synthesis in bacteria 1. In addition, it has been shown that in the case of at least 2 amino acids, their participation in RNA control was dependent on a functional aminoacyl-tRNA synthetase *,a. However, it was not apparent from these studies whether the formation of the amino acid-adenylate enzyme complex or of aminoacyl-tRNA was the necessary step for regulation of RNA synthesis. We now report evidence for the participation of valine tRNA in the control of RNA synthesis in Escherichia coli W strain M42-II, an isoleucine-valine auxotroph stringent for RNA control. For these experiments, DL-x-aminobutyrate and DL-threo~-amino-fl-chlorobutyrate (chlorobutyrate) were used. These valine analogues are activated by valyl-tRNA synthetase 4 (L-valine :tRNA ligase (AMP), EC 6.1.1.9) but only chlorobutyrate is transferred to valine tRNA 5. The cells were grown at 37 ° with aeration in minimal medium e supplemented with L-isoleucine (40 #g/ml), L-valine (80/~g/ml), L-phenylalanine (20/zg/ml), uracil (20 #g/ml), sodium acetate (0.2 %) and glucose (0.2 %). The cells were harvested at a density equivalent to 15o K l e t t Summerson colorimeter units, washed twice with minimal medium, and resuspended in minimal medium supplemented with L-isoleucine (4° #g/ml). The cells were placed in flasks at a density equal to 40 Klett-Summerson colorimeter units. L-Valine (4° #g/ml) was added to one set of flasks, a second set received no valine, and chlorobutyrate (60 #g/ml) and aminobutyrate (30o/~g/ml) were added to a third and fourth set of flasks, respectively. RNA and protein synthesis was measured by the uptake of E14Clphenylalanine and [14C]uracil into trichloroacetic acid-insoluble material 5. As expected, RNA and protein synthesis were completely blocked when the auxotroph was deprived of valine (Fig. I). However, the addition of chlorobutyrate to the valine-starved culture resulted in considerable synthesis of RNA while protein synthesis was essentially blocked. The analogue caused approximately a 5-fold uncoupling of RNA and protein synthesis. In contrast, no RNA or protein synthesis TABLE
I
CHEMICAL DETERMINATION OF R N A AND PROTEIN SYNTHESIZED UPON ADDITION OF VALINE AND CHLOROBUTYRATE IN E . coli STRAIN M 4 2 - I I The cells were grown and harvested as described in the text. They were resuspended in minimal m e d i u m w i t h L - i s o l e u c i n e (4 ° / , g / m l ) a n d L - v a l i n e (4 ° / z g / m l ) o r c h l o r o b u t y r a t e ( 6 0 / z g / m l ) . S a m ples were removed and chemical determinations were made for protein s and RNA*. The values for RNA and protein were normalized to the values obtained at zero time.
Time after addition (min)
Compound added
RNA
o 3° 60 o 3o 60
Valine Valine Valine Chlorobutyrate Chlorobutyrate Chlorobutyrate
i.oo 1.3o 2.5 ° I.oo I.I4 1.71
Protein
RNA Protein
i.oo 1.34 2. I8 I.OO I.OO i.oo
i.oo 0.97 I.iO I.OO I.I4 1.71
BiocMm. Biophys. Acta, 179 ( 1 9 6 9 ) 5 1 5 - 5 1 7
516
PRELIMINARY NOTES
I000
I
I
I
I
~ooo
I
I / 6
/
900 --
F
c E ~-- 7 0 0
o
•
z
-
~
w
500
-
~
X~
Q: a:
o
300
300
lO(
o
15
30 INCUBATION
45 TIME (MIN)
60
o
J
I
15 30 INCUBATION TIME
f
45
60
(giN)
Fig. I. Effect of ~-aminobutyrate and chlorobutyrate on RNA and protein synthesis. The cells were harvested during exponential growth and resuspended in minimal medium with supplementation as described in the text. RNA and protein synthesis was followed by the uptake of [14CJuracil (15 #g/ml) and DL-[14C]phenylalanine (3°/zg/ml). To each flask containing [t4CJuracil was added DL-[x2C~phenylalanine (3o/zg/ml) and the flasks with E14C~phenylalanine received [z2Cluracil (15/~g/ml). Samples were removed and the radioactivity incorporated into trichloroacetic acid-insoluble material measured as described previouslys. Valine excess: C), RNA; 0 , protein. No valine: &, RNA; A, l~rotein. No valine plus ct-aminobutyrate: ~ , RNA; II, protein. No valine plus chlorobutyrate: ×, RNA; ®, protein. Fig. 2. Effect of valine starvation and chlorobutyrate on lipid synthesis. The cells were harvested during exponential growth and resuspended in minimal medium plus ~]4C]acetate (o.2 %) and other supplements as described in the text. Lipid synthesis was measured by the incorporation of [x4C~acetate as determined by the method of SOKA'vVAet aLL 0 , valine (4o/tg/m]); A, no valine or no valine plus ~-aminobutyrate (3oo/*g/ml); ×, no valine plus chlorobutyrate (60 btg/ml).
occurred when aminobutyrate was added to the cultures starved of valine. To test the validity of these isotope incorporation studies, chemical determinations of RNA and protein were performed on samples from cultures grown as described in the previous experiment. As shown in Table I, the addition of chlorobutyrate to a culture deprived of valine resulted in a 7 ° % increase in the RNA to protein ratio. It has recently been suggested that the synthesis of lipids in E. coli is regulated by amino acids in a similar manner to RNAL To test this hypothesis, lipid synthesis was followed in strain M42-II grown in the absence of valine, with and without chlorobutyrate. Lipid synthesis was measured by the incorporation of E14C~acetate as described previously 7 except that the radioactivity of the samples was determined with a Packard scintillation counter. The data in Fig. 2 show that [14C~acetate incorporation was essentially blocked during valine starvation. Significantly, the addition of chlorobutyrate allowed for appreciable lipid formation in the absence of valine. Biochim. Biophys. Acta, 179 (1969) 515-517
517
PRELIMINARY NOTES
The results reported here show that although x-aminobutyrate and chlorobutyrate are activated by valyl-tRNA synthetase, only the analogue which can attach to valine tRNA can support RNA synthesis without the concomitant synthesis of protein. These observations confirm the earlier report a that valine must be activated in order to participate in RNA control and they further suggest that valine must be transferred to tRNA before it can support RNA synthesis. The data further indicate that tRNA may also be involved in the postulated regulation of lipid biosynthesis by amino acidsL We thank M. Rabinovitz for chlorobutyrate. L. S. W. is a Postdoctoral fellow of the American Cancer Society. This work was supported by a grant from the Joint Awards Council of the State University of New York.
Biochemistry Section, Department o/ Biological Sciences, State University of New York, Stony Brook, N. Y. (U.S.A.)
L . S . WILLIAMS M. FREUNDLICH
z G. EDLIN AND P. BRODA, Bacteriol. Rev., 32 (1968) 206. 2 W. L. FANGMAN AND F. C. •EIDHARDT, J. Biol. Chem., 239 (1964) 1844. 3 L. EIDLIC AND F. C. NEIDIIARDT, J. Bacteriol., 89 (1965) 706. 4 F. B. BERGMANN, P. BERG AND M. DIECKMANN, J. Biol. Chem., 236 (1961) 1735. 5 M. FREUNDLICrI, Science, 157 (1967) 823. 6 B. D. DAVIS AND E. S. MII~GIOLI, J. Bacteriol., 6o (195o) 17. 7 Y. SOKAWA, E. NAKAO AND Y. KAZlRO, Biochem. Biophys. Res. Commun., 33 (1968) lO8. 8 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 9 A. MARSHAK AND H. J. VOGEL, J. Biol. Chem., I89 (1951) 597.
Received January I7th, I969 Biochim. Biophys, Acta, I79 (I969) 515-517
BBA 91244 The presence of 2-methylthio-N6.(A2.isopentenyl)adenosine phenylalanine tronsfer RNA's from Escherichia coli
in serine ond
We previously reported the characterization of a new modified nucleoside in Escherichia coli tRNA Tyr as 2-methylthio-N6-isopentenyladenosine1. It is of particular interest that this nucleoside is located next to the 3'-end of the anticodon of E. coli tRNA Tyr (ref, 2). This nucleoside was also independently isolated and identified as one of the cytokinin active components from the total mixture of E. coli tRNA by BURROWS et al. 3. The presence of N6-isopentenyladenosine in exactly the same position (next to the anticodon) of yeast tRNA Tyr (ref. 4) and tRNA set (ref. 5) raised the question of whether or not E. coli tRNA set as well as other species of tRNA from E. coli contains 2-methylthio-Ne-isopentenyladenosine. This paper briefly reports the identification of 2-methylthio-N*-isopentenyladenosine in tRNA s~r and tRNA Ph~ from E. coll. E. coli tRNA Ph~ was prepared as described previously s. E. coli tRNA se~ was first fractionated into three species (tRNA s~ and tRNAS~ r for codons of UC series, Biochim, Biophys. Acta, 179 (1969) 517-52o