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Catabolite repression and the induction of β-galactosidase
BIOCHIMICA ET BIOPHYSICA ACTA
835
Preliminary Notes PN 6073 Catabolite repression and the induction of B-galactosidose The initial kinetics of the induced synthesis of /~-galactosidase (EC 3.2.1.23) in Escherichia coli have been studied by ~PARDEE AND PRESTIDGE1. They showed that after the addition of the inducer 3-5 rain elapse before the first appearance of the enzyme; if the inducer is removed at the end of this period of lag, enzyme synthesis proceeds for a period of approx. 8 rain. BUSSARD et al. 2 have reported that 5-fluorouracil, which is readily incorporated into unstable messenger RNA 3, inhibits the constitutive synthesis of/5-galactosidase and brings about the formation of an enzymically inactive protein serologically related to /5-galactosidase. The inhibitory effect of 5-fluorouraeil on the induction of/5-galactosidase becomes apparent 4-5 rain after its addition 1. These observations are in agreement with the hypothesis of JACOB AND MONOD4 that the inducer stimulates the formation of an unstable messenger RNA which can subsequently in the absence of the inducer direct the formation of/%galactosidase. The experiments shown in Fig. IA, in which inducer was allowed to act for only 4 rain and was then removed by filtration, reveal that 5-fluorouracil inhibits the synthesis of/%galactosidase only when it is present during the 4-rain period of induction; its presence during the 8-rain period following the removal of inducer affects neither the capacity of the cells to synthesize/~-galactosidase nor the exponential decline of this capacity that is observed following removal of the inducer (Fig. ID). The small amount of enzyme which is formed in cells induced in the presence of 5-fluorouracil is accompanied by an amount of the enzymically inactive protein (determined by the method of HAMERS AND HAMERS-CASTERMAN1) equivalent to about one-third of the enzyme. None of this protein is formed by cells which after normal induction produce/%galactosidase in the presence of 5-fluorouracil. The altered protein synthesized by the cells presumably reflects the incorporation of 5-fluorouracil into messenger RNA during induction; such an incorporation was demonstrated b y t h e use of [m~cl-5fluorouracil. It has been shown previously that the synthesis of/~-galactosidase is repressed by catabolites in cells which are supplied with a source of energy but deprived of an essential building block 6. It could now be shown that the catabolites affect the induction of the enzyme rather than its production. When a threonine-requiring mutant of E . coli is induced in a medium containing the energy source glycerol but lacking threonine, it fails to produce /~-galactosidase subsequently in an inducer-free medium containing both glycerol and threonine (Fig. IB). On the other hand, induction in a medium deprived of both glycerol and threonine permits the subsequent synthesis of /~-galactosidase without inducer not only in media containing both glycerol and threonine (Fig. IB), or lacking both glycerol and threonine (Fig. IC), but also in a medium containing glycerol but no threonine (Fig. IC). Similar results are obtained when the cells are made tryptophan deficient by the addition of 5-methyltryptophan. Furthermore, in the absence of an energy source the enzyme can be Biochim. Biophys..4eta, 6x (r96z) 835-837
836
PRELIMINARY NOTES
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Fig. I. fl-Galactosidase s y n t h e s i s a f t e r t h e r e m o v a l of inducer. T h e b a c t e r i a l c u l t u r e w a s IK-I2, s t r a i n 2ooP, a f l - g a l a c t o s i d e - p e r m e a s e - n e g a t i v e m u t a n t (i + o + z + y - ) w h i c h r e q u i r e s t h r e o n i n e , leucine, a n d t h i a m i n e (from Dr. J. MOXOD'S collection; o b t a i n e d f r o m Dr. S. E. LURIA). Cell~ g r o w i n g e x p o n e n t i a l l y a t 3 °0 in a m e d i u m s c o n t a i n i n g glycerol (0.2 %), I.-threonine (2. lO -4 M), L-leucine (2" IO-* M) a n d t h i a m i n e (0. 5/zg/ml) were w a s h e d a n d r e s u s p e n d e d in m i n e r a l - s a l t s m e d i a w i t h v a r i o u s s u p p l e m e n t s . A f t e r 5 rain t h e i n d u c e r isopropylthio-fl-D-galactoside (5" lO-4 M) w a s a d d e d a n d t h e i n c u b a t i o n w a s c o n t i n u e d for 4 m i n a t 3 °° w i t h s h a k i n g . A t t h i s t i m e t h e ind u c e r w a s q u i c k l y r e m o v e d b y f i l t r a t i o n t h r o u g h a m e m b r a n e filter (SS, A-coarse, 5 ° m m ) a n d t h e b a c t e r i a were w a s h e d w i t h cold m e d i u m free of inducer, s u p p l e m e n t s , a n d sources of c a r b o n a n d nitrogen. T h e b a c t e r i a were s u s p e n d e d in p r e - w a r m e d m e d i u m free of i n d u c e r b u t c o n t a i n i n g L-[I-]iC]leucine (specific a c t i v i t y 2 5 . 4 m C / m m o l e , final c o n c e n t r a t i o n o.o5/~C/ml ) a n d t h e s u p p l e m e n t s i n d i c a t e d b e l o w a n d were i n c u b a t e d a t 3 o°. S a m p l e s were t a k e n for a s s a y of fl-galact o s i d a s e a c t i v i t y 3 a n d for m e a s u r e m e n t of [14C]leucine i n c o r p o r a t i o n into cellular protein. A correction w a s m a d e for i n h i b i t i o n of e n z y m e a c t i v i t y b y isopropylthio-~-I)-galactoside. T h e e n z y m e a c t i v i t y of s a m p l e s t a k e n f r o m m e d i a w h i c h did n o t c o n t a i n glycerol w a s also corrected since it w a s f o u n d t h a t t h e a s s a y g a v e s l i g h t l y h i g h e r v a l u e s w h e n carried o u t in t h e presence of glycerol. I n Fig. IA, B, a n d C, t h e i n c r e a s e s in fl-galactosidase (solid lines) a n d t h e i n c r e a s e s in protein (broken lines) a r e s h o w n . I n Fig. ID, t h e differential r a t e of e n z y m e s y n t h e s i s A units fl-galactosidase/min A c o u n t s [14C]leucine i n c o r p o r a t e d / r a i n
X
lO 3
is p l o t t e d a g a i n s t t i m e . A r r o w s indicate t h e differential r a t e of fl-galactosidase s y n t h e s i s in t h e p r e s e n c e of t h e i n d u c e r a f t e r t h e s t e a d y s t a t e h a s b e e n reached. Solid arrow: in a m e d i u m d e p r i v e d of b o t h glycerol a n d t h r e o n i n e ; b r o k e n arrow: in a c o m p l e t e m e d i u m . O - O , i n d u c e d in a c o m p l e t e m e d i u m ; t h e n s u s p e n d e d in a c o m p l e t e m e d i u m a f t e r r e m o v a l of t h e inducer, rT-rN, s a m e as O - O , b u t w i t h 5-fluorouracil (20/~g/ml) p r e s e n t o n l y d u r i n g t h e period of i n d u c t i o n . ZX-ZX, s a m e as O - O b u t w i t h 5-fluorouracil p r e s e n t o n l y a f t e r r e m o v a l of t h e inducer. O - O , i n d u c e d in a m e d i u m w i t h o u t glycerol a n d t h r e o n i n e ; t h e n s u s p e n d e d in c o m p l e t e m e d i u m a f t e r r e m o v a l of t h e inducer. V - V , i n d u c e d in a m e d i u m w i t h glycerol b u t w i t h o u t t h r e o n i n e ; t h e n s u s p e n d e d in c o m p l e t e m e d i u m a f t e r r e m o v a l of t h e inducer. A - A , s a m e as 0 - 0 , b u t s u s p e n d e d in a m e d i u m w i t h glycerol a n d w i t h o u t t h r e o n i n e . I - i , s a m e as O - Q , b u t s u s p e n d e d in a m e d i u m l a c k i n g b o t h glycerol a n d t h r e o n i n e .
induced even when all measurable protein synthesis has been inhibited by chloramphenicol. After removal of the inducer the differential rate of fl-galactosidase synthesis, which is a measure of the capacity of the cells for synthesis of the enzyme relative to total protein synthesis, declines exponentially (Fig. ID) ; the half life of this enzymeforming capacity is approx. 2.5 rain at 3 °o irrespective of the rate of protein synthesis. Biochim. Biophys. Acta, 61 (1962) 835-837
PRELIMINARY NOTES
837
The capacity for ~-glactosidase synthesis of E. coli appears to have a stability somewhat comparable to the stability of Bacillus subtilis messenger RNA whose half life at 33.5 ° has been estimated by LEVINTHALet al. v to be 1.4 min. The initial differential rate of fl-galactosidase synthesis, obtained by extrapolation of the decay curve (Fig. ID), is also independent of the overall rate of protein synthesis and approximately equals the differential rate at which the cells produce the enzyme in the presence of the inducer after the steady state has been reached. These results provide strong evidence for the concept that the inducer stimulates the formation of an unstable messenger RNA specific for fl-galactosidase and that the catabolite repressor inhibits the formation of this specific RNA fraction. This work was supported in part by grants from the U.S. Public Health Service (CY-52IO and RG-7446) and the National Science Foundation (G-Io946). We are indebted to Dr. C. LEVINTHAL for valuable discussions.
Department o/ Biology, Massachusetts Institute o[ Technology, Cambridge, Mass. (U.S.A.)
DAISUKE NAKADA BORIS MAGASANIK
1 A. B. PARDEE AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 49 (1961) 77. 2 ~k. BUSSARD, S. NAONO, F. GROS AND J. MONOD, Compt. Rend. 25o (196o) 4049 . 3 F. GROS, N. GILBERT, H. H. HIATT, G. ATTARDI, 1~. F. SPAHR AND J. D. WATSON, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) I I i . 4 F. JACOB AND J. MONOD, J. Mol. Biol., 3 (1961) 318. 5 R. HAMERS AND C. HAMERS-CASTERMAN, J. Mol. Biol., 3 (1961) 166. ]3. MAGASANIK, Cold Spring Harbor Symp. Quant. Biol., 26 (1961) 249. C. LEVINTHAL, A. KEYNAN AND A.. HIGA, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1631. 8 D. I~AKADA, Biochim. Biophys. Acta, 55 (1962) 505 . 9 A. ]~. PARDEE, F. JACOB AND J. MONOD, J. Mol. Biol., i (1959) 165. x0 L. A. HERZENBERG, Biochim. Biophys. Acta, 31 (1959) 525 .
Received October I5th, 1962 Biochim. Biophys. Acta, 61 (1962) 835-837
835
Preliminary Notes PN 6073 Catabolite repression and the induction of B-galactosidose The initial kinetics of the induced synthesis of /~-galactosidase (EC 3.2.1.23) in Escherichia coli have been studied by ~PARDEE AND PRESTIDGE1. They showed that after the addition of the inducer 3-5 rain elapse before the first appearance of the enzyme; if the inducer is removed at the end of this period of lag, enzyme synthesis proceeds for a period of approx. 8 rain. BUSSARD et al. 2 have reported that 5-fluorouracil, which is readily incorporated into unstable messenger RNA 3, inhibits the constitutive synthesis of/5-galactosidase and brings about the formation of an enzymically inactive protein serologically related to /5-galactosidase. The inhibitory effect of 5-fluorouraeil on the induction of/5-galactosidase becomes apparent 4-5 rain after its addition 1. These observations are in agreement with the hypothesis of JACOB AND MONOD4 that the inducer stimulates the formation of an unstable messenger RNA which can subsequently in the absence of the inducer direct the formation of/%galactosidase. The experiments shown in Fig. IA, in which inducer was allowed to act for only 4 rain and was then removed by filtration, reveal that 5-fluorouracil inhibits the synthesis of/%galactosidase only when it is present during the 4-rain period of induction; its presence during the 8-rain period following the removal of inducer affects neither the capacity of the cells to synthesize/~-galactosidase nor the exponential decline of this capacity that is observed following removal of the inducer (Fig. ID). The small amount of enzyme which is formed in cells induced in the presence of 5-fluorouracil is accompanied by an amount of the enzymically inactive protein (determined by the method of HAMERS AND HAMERS-CASTERMAN1) equivalent to about one-third of the enzyme. None of this protein is formed by cells which after normal induction produce/%galactosidase in the presence of 5-fluorouracil. The altered protein synthesized by the cells presumably reflects the incorporation of 5-fluorouracil into messenger RNA during induction; such an incorporation was demonstrated b y t h e use of [m~cl-5fluorouracil. It has been shown previously that the synthesis of/~-galactosidase is repressed by catabolites in cells which are supplied with a source of energy but deprived of an essential building block 6. It could now be shown that the catabolites affect the induction of the enzyme rather than its production. When a threonine-requiring mutant of E . coli is induced in a medium containing the energy source glycerol but lacking threonine, it fails to produce /~-galactosidase subsequently in an inducer-free medium containing both glycerol and threonine (Fig. IB). On the other hand, induction in a medium deprived of both glycerol and threonine permits the subsequent synthesis of /~-galactosidase without inducer not only in media containing both glycerol and threonine (Fig. IB), or lacking both glycerol and threonine (Fig. IC), but also in a medium containing glycerol but no threonine (Fig. IC). Similar results are obtained when the cells are made tryptophan deficient by the addition of 5-methyltryptophan. Furthermore, in the absence of an energy source the enzyme can be Biochim. Biophys..4eta, 6x (r96z) 835-837
836
PRELIMINARY NOTES
I
do
I I
c
i
I
:
/
4
6
D
~IOIC
!
\
~_
~
0 0 0 ',
~ ~c
Z
uJ
Ld
..
L,J 2.C
(,9 0
500 o <
,I(.9
// ~o i'o / .~'~-V~ " -.~ -
~_
-
,,z, t.c
a_
a
_
0
i
I
5
I0
0
5 TIME
I0
5
I0
0
0
5
_
_ TIME
(MIN)
8
(MIN)
Fig. I. fl-Galactosidase s y n t h e s i s a f t e r t h e r e m o v a l of inducer. T h e b a c t e r i a l c u l t u r e w a s IK-I2, s t r a i n 2ooP, a f l - g a l a c t o s i d e - p e r m e a s e - n e g a t i v e m u t a n t (i + o + z + y - ) w h i c h r e q u i r e s t h r e o n i n e , leucine, a n d t h i a m i n e (from Dr. J. MOXOD'S collection; o b t a i n e d f r o m Dr. S. E. LURIA). Cell~ g r o w i n g e x p o n e n t i a l l y a t 3 °0 in a m e d i u m s c o n t a i n i n g glycerol (0.2 %), I.-threonine (2. lO -4 M), L-leucine (2" IO-* M) a n d t h i a m i n e (0. 5/zg/ml) were w a s h e d a n d r e s u s p e n d e d in m i n e r a l - s a l t s m e d i a w i t h v a r i o u s s u p p l e m e n t s . A f t e r 5 rain t h e i n d u c e r isopropylthio-fl-D-galactoside (5" lO-4 M) w a s a d d e d a n d t h e i n c u b a t i o n w a s c o n t i n u e d for 4 m i n a t 3 °° w i t h s h a k i n g . A t t h i s t i m e t h e ind u c e r w a s q u i c k l y r e m o v e d b y f i l t r a t i o n t h r o u g h a m e m b r a n e filter (SS, A-coarse, 5 ° m m ) a n d t h e b a c t e r i a were w a s h e d w i t h cold m e d i u m free of inducer, s u p p l e m e n t s , a n d sources of c a r b o n a n d nitrogen. T h e b a c t e r i a were s u s p e n d e d in p r e - w a r m e d m e d i u m free of i n d u c e r b u t c o n t a i n i n g L-[I-]iC]leucine (specific a c t i v i t y 2 5 . 4 m C / m m o l e , final c o n c e n t r a t i o n o.o5/~C/ml ) a n d t h e s u p p l e m e n t s i n d i c a t e d b e l o w a n d were i n c u b a t e d a t 3 o°. S a m p l e s were t a k e n for a s s a y of fl-galact o s i d a s e a c t i v i t y 3 a n d for m e a s u r e m e n t of [14C]leucine i n c o r p o r a t i o n into cellular protein. A correction w a s m a d e for i n h i b i t i o n of e n z y m e a c t i v i t y b y isopropylthio-~-I)-galactoside. T h e e n z y m e a c t i v i t y of s a m p l e s t a k e n f r o m m e d i a w h i c h did n o t c o n t a i n glycerol w a s also corrected since it w a s f o u n d t h a t t h e a s s a y g a v e s l i g h t l y h i g h e r v a l u e s w h e n carried o u t in t h e presence of glycerol. I n Fig. IA, B, a n d C, t h e i n c r e a s e s in fl-galactosidase (solid lines) a n d t h e i n c r e a s e s in protein (broken lines) a r e s h o w n . I n Fig. ID, t h e differential r a t e of e n z y m e s y n t h e s i s A units fl-galactosidase/min A c o u n t s [14C]leucine i n c o r p o r a t e d / r a i n
X
lO 3
is p l o t t e d a g a i n s t t i m e . A r r o w s indicate t h e differential r a t e of fl-galactosidase s y n t h e s i s in t h e p r e s e n c e of t h e i n d u c e r a f t e r t h e s t e a d y s t a t e h a s b e e n reached. Solid arrow: in a m e d i u m d e p r i v e d of b o t h glycerol a n d t h r e o n i n e ; b r o k e n arrow: in a c o m p l e t e m e d i u m . O - O , i n d u c e d in a c o m p l e t e m e d i u m ; t h e n s u s p e n d e d in a c o m p l e t e m e d i u m a f t e r r e m o v a l of t h e inducer, rT-rN, s a m e as O - O , b u t w i t h 5-fluorouracil (20/~g/ml) p r e s e n t o n l y d u r i n g t h e period of i n d u c t i o n . ZX-ZX, s a m e as O - O b u t w i t h 5-fluorouracil p r e s e n t o n l y a f t e r r e m o v a l of t h e inducer. O - O , i n d u c e d in a m e d i u m w i t h o u t glycerol a n d t h r e o n i n e ; t h e n s u s p e n d e d in c o m p l e t e m e d i u m a f t e r r e m o v a l of t h e inducer. V - V , i n d u c e d in a m e d i u m w i t h glycerol b u t w i t h o u t t h r e o n i n e ; t h e n s u s p e n d e d in c o m p l e t e m e d i u m a f t e r r e m o v a l of t h e inducer. A - A , s a m e as 0 - 0 , b u t s u s p e n d e d in a m e d i u m w i t h glycerol a n d w i t h o u t t h r e o n i n e . I - i , s a m e as O - Q , b u t s u s p e n d e d in a m e d i u m l a c k i n g b o t h glycerol a n d t h r e o n i n e .
induced even when all measurable protein synthesis has been inhibited by chloramphenicol. After removal of the inducer the differential rate of fl-galactosidase synthesis, which is a measure of the capacity of the cells for synthesis of the enzyme relative to total protein synthesis, declines exponentially (Fig. ID) ; the half life of this enzymeforming capacity is approx. 2.5 rain at 3 °o irrespective of the rate of protein synthesis. Biochim. Biophys. Acta, 61 (1962) 835-837
PRELIMINARY NOTES
837
The capacity for ~-glactosidase synthesis of E. coli appears to have a stability somewhat comparable to the stability of Bacillus subtilis messenger RNA whose half life at 33.5 ° has been estimated by LEVINTHALet al. v to be 1.4 min. The initial differential rate of fl-galactosidase synthesis, obtained by extrapolation of the decay curve (Fig. ID), is also independent of the overall rate of protein synthesis and approximately equals the differential rate at which the cells produce the enzyme in the presence of the inducer after the steady state has been reached. These results provide strong evidence for the concept that the inducer stimulates the formation of an unstable messenger RNA specific for fl-galactosidase and that the catabolite repressor inhibits the formation of this specific RNA fraction. This work was supported in part by grants from the U.S. Public Health Service (CY-52IO and RG-7446) and the National Science Foundation (G-Io946). We are indebted to Dr. C. LEVINTHAL for valuable discussions.
Department o/ Biology, Massachusetts Institute o[ Technology, Cambridge, Mass. (U.S.A.)
DAISUKE NAKADA BORIS MAGASANIK
1 A. B. PARDEE AND L. S. PRESTIDGE, Biochim. Biophys. Acta, 49 (1961) 77. 2 ~k. BUSSARD, S. NAONO, F. GROS AND J. MONOD, Compt. Rend. 25o (196o) 4049 . 3 F. GROS, N. GILBERT, H. H. HIATT, G. ATTARDI, 1~. F. SPAHR AND J. D. WATSON, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) I I i . 4 F. JACOB AND J. MONOD, J. Mol. Biol., 3 (1961) 318. 5 R. HAMERS AND C. HAMERS-CASTERMAN, J. Mol. Biol., 3 (1961) 166. ]3. MAGASANIK, Cold Spring Harbor Symp. Quant. Biol., 26 (1961) 249. C. LEVINTHAL, A. KEYNAN AND A.. HIGA, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1631. 8 D. I~AKADA, Biochim. Biophys. Acta, 55 (1962) 505 . 9 A. ]~. PARDEE, F. JACOB AND J. MONOD, J. Mol. Biol., i (1959) 165. x0 L. A. HERZENBERG, Biochim. Biophys. Acta, 31 (1959) 525 .
Received October I5th, 1962 Biochim. Biophys. Acta, 61 (1962) 835-837