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 PRESTIDGE 1. They showed that after the addition of the inducer 3-5 rain elapse before the first appearance of the en- zyme; 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-fluoroura- cil, which is readily incorporated into unstable messenger RNA 3, inhibits the consti- tutive synthesis of/5-galactosidase and brings about the formation of an enzymical- ly 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 MONOD 4 that the inducer stimulates the formation of an unstable messenger RNA which can subsequently in the absence of the inducer direct the formation of/%galac- tosidase.

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 syn- thesis 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-CASTERMAN 1) equivalent to about one-third of the enzyme. None of this protein is formed by cells which after normal induction pro- duce/%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 by the use of [m~cl-5- fluorouracil.

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. I t could now be shown that the catabolites affect the in- duction of the enzyme rather than its production. When a threonine-requiring mu- tant of E. coli is induced in a medium containing the energy source glycerol but lack- ing threonine, it fails to produce /~-galactosidase subsequently in an inducer-free medium containing both glycerol and threonine (Fig. IB). On the other hand, in- duction 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-me- thyltryptophan. 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. f l -Galactos idase s y n t h e s i s a f t e r t he r e m o v a l of inducer . The bac te r ia l cu l tu re was IK-I2, s t r a in 2ooP, a f l -ga lac tos ide -permease -nega t ive m u t a n t (i + o + z + y - ) wh ich requi res th reon ine , leucine, and t h i a m i n e (from Dr. J. MOXOD'S collection; ob ta ined f rom Dr. S. E. LURIA). Cell~ g rowing exponen t i a l l y a t 3 °0 in a m e d i u m s con ta in ing glycerol (0.2 %), I . - threonine (2. lO -4 M), L-leucine (2" IO-* M) and t h i a m i n e (0. 5 /zg/ml) were washed and r e suspended in m ine ra l - s a l t s m e d i a w i th va r ious supp l emen t s . Af te r 5 rain t he inducer i sopropyl th io-f l -D-galac tos ide (5" lO-4 M) was added a n d t he i ncuba t i on was con t inued for 4 m i n a t 3 °° w i th shaking . At th i s t ime t he in- ducer was qu ick ly r e m o v e d by f i l t r a t ion t h r o u g h a m e m b r a n e f i l ter (SS, A-coarse, 5 ° m m ) a n d t he bac t e r i a were w a s h e d wi th 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 ca rbon and n i t rogen. The bac 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 inducer b u t con ta in ing L-[I-] iC]leucine (specific a c t i v i t y 2 5 . 4 m C / m m o l e , f inal concen t r a t i on o.o5/~C/ml ) and t he s u p p l e m e n t s ind ica ted be low and were i ncuba t ed a t 3 o°. Samples were t aken for a s s a y of fl-galac- tos idase ac t iv i ty 3 and for m e a s u r e m e n t of [14C]leucine incorpora t ion in to cellular protein. A correct ion was m a d e for inh ib i t ion of e n z y m e ac t i v i t y by isopropyl thio-~-I)-galactoside. The e n z y m e ac t iv i ty of s amp le s t a k e n f rom med i a which did n o t con ta in glycerol was also corrected s ince i t was found t h a t t he a s s a y gave s l igh t ly h ighe r va lues when carr ied o u t in t he presence of glycerol.

In Fig. IA, B, a n d C, t he increases in f l -galactos idase (solid lines) and the increases in pro- te in (broken lines) a re shown. I n Fig. ID, t he d i f ferent ia l ra te of e n z y m e syn the s i s

A un i t s f l -ga lac tos idase /min X l O 3

A c o u n t s [14C]leucine incorpora ted / ra in

is p lo t t ed aga in s t t ime . Arrows indica te the d i f ferent ia l ra te of f l -galactosidase syn the s i s in t he p resence of t he inducer a f t e r the s t e a d y s t a t e ha s been reached. Solid arrow: in a m e d i u m depr ived of b o t h glycerol a n d th reon ine ; b roken arrow: in a comple te m e d i u m . O - O , induced in a comple t e m e d i u m ; t h e n s u s p e n d e d in a comple t e m e d i u m af te r r e m o v a l of t he inducer , rT-rN, s a m e as O - O , b u t w i th 5-f luorouraci l (20/~g/ml) p r e s e n t on ly du r i ng t he per iod of induc t ion . ZX-ZX, s ame as O - O b u t wi th 5-f luorouraci l p r e sen t on ly a f te r r e m o v a l of t he inducer . O - O , induced in a m e d i u m w i t h o u t glycerol a n d th reon ine ; t h e n su spended in comple te m e d i u m af te r r e m o v a l of the inducer . V - V , i nduced in a m e d i u m wi th glycerol b u t w i t h o u t th reon ine ; t h e n su spended in comple te m e d i u m af te r r e m o v a l of t he inducer . A - A , s ame as 0 - 0 , b u t su spended in a m e d i u m wi th gly- cerol and w i t h o u t th reon ine . I - i , s ame as O - Q , b u t su spended in a m e d i u m lacking bo th

glycerol and threonine .

induced even when all measurable protein synthesis has been inhibited by chloram- phenicol.

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 enzyme- forming 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 some- what comparable to the stability of Bacillus subtilis messenger RNA whose half life at 33.5 ° has been estimated by LEVINTHAL et 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 approxi- mately equals the differential rate at which the cells produce the enzyme in the pres- ence 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