The stability of ribosomes in uracil-starved Escherichia coli

414

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 8127

THE STABILITY

OF RIBOSOMES IN URACIL-STARVED ESCHERICHIA COLI

DAISUKE

N A K A D A * AND I S S A R S M I T H ' *

Department o/ Zoology, Columbia University, New York, N.Y. and Department o[ Biology, Massachusetts Institute o/ Technology, Cambridge, Mass. (U.S.A.) (Received M a r c h 9th, I962)

SUMMARY

The effect of uracil starvation and of methionine starvation on the synthesis of ribosomal RNA and protein in Fscherichia colt was investigated. The study depended on the separation of ~SN-containing from ~4N-containing ribosomes in a CsC1 density gradient, and on the separation of ribosomes from messenger RNA in a sucrose density gradient. It was found that under these conditions of starvation neither synthesis of new ribosomes nor turnover of ribosomal RNA and protein occurs. On the other hand, the turnover of messenger RNA continues during uracil starvation.

INTRODUCTION

During growth the ribosomal RNA molecules of Escherichia colt have been shown to be stable 1. However, under conditions of amino acid or nitrogen starvation when no net synthesis of protein and RNA occurs, considerable incorporation of exogenous protein and RNA precursors into ribosomal protein and RNA has been reported ~,3. Under conditions of uracil starvation, there is no net synthesis of RNA and little net synthesis of protein. It seemed of interest to investigate the fate of the ribosomes in such uracil-starved cells: either the ribosomes are stable, or there is partial turnover of the ribosomal protein and RNA, or there occurs complete destruction and reformation of the whole ribosomes. To distinguish between these possibilities, 14N-containing ribosomes were separated from 15N-containing ribosomes by a modification of the method of BRENNER et al. 4. MATERIALS AND METHODS

Bacteria A uracil-less m u t a n t of E. colt, 63-86, was used throughout the experiments. Method o/cultivation and uracil starvation Bacteria were grown to a cell density of 4" IOS/ml in a mineral salts-glycerol medium 5 which contained 15NH4C1 instead of 14NH4C1 and was supplemented with A b b r e v i a t i o n : TCA, thrichloroacetic acid. * P r e s e n t a d d r e s s : D e p a r t m e n t of Biology, M a s s a c h u s e t t s I n s t i t u t e of T e c h n o l o g y , C a m b r i d g e , Mass. (U.S.A.). ** P r e s e n t a d d r e s s : D e p a r t m e n t of Microbiology, N e w Y o r k U n i v e r s i t y , N e w York, N.Y.

(U.S.A.). Biochim. Biophys. Acta, 61 (I962) 4 1 4 - 4 2 o

S T A B I L I T Y OF R I B O S O M E S D U R I N G S T A R V A T I O N

415 .

o.oooi M uracil; a small inoculum was used so that the bacteria were fully 15N-labelled. The cells were harvested, washed twice by centrifugation and were resuspended to the original volume with 14N-medium lacking uracil. These suspensions were incubated at 37 ° with shaking for 3o min to allow the phase of rapid residual synthesis of protein to come to an end. At this time, [8-14C]adenine (o.I/tC/ml) or L-[I-1lC]leucine (o.I pC/ml) was added and the uracil starvation was continued. During 60 min of uracil starvation in the presence of either radioisotope, there was no detectable net increase of RNA and of protein. For the preparation of crude extracts which were used for the sucrose density gradient centrifugation studies, bacteria were grown in a 14N-medium containing uracil and, after washing, were transferred to a 14N-medium lacking uracil.

Preparation o/ crude extracts and puri/ied ribosomes Aliquots were removed from the uracil starvation medium at timed intervals after the addition of radioisotope and poured over an equal volume of crushed ice made by freezing a Tris-Mg buffer (0.005 M Tris, o.oi M magnesium acetate, p H 7-3) containing o.oi M sodium azide, this immediately stopped the incorporation of radioisotope. All the following procedures were carried out at 4 ° and the buffer used throughout was 0.005 M Tris containing o.oi M magnesium acetate, pH 7.3. After extensive washing with buffer, the cells were ground with 2 vol. of alumina for 5 rain and extracted with a small volume of buffer. The extracts were freed of alumina and debris by two centrifugations at 20 ooo × g for 20 rain. These crude extracts were then subjected to centrifugation at lO5 o o o x g for 9° min. The resulting pellets, which consisted of crude ribosomes, were resuspended with butfer to the original volume and again centrifuged at lO5 ooo × g for 9° min. The sedimented ribosomes were resuspended with buffer and the suspension was centrifuged at 20 ooo x g for 20 min. Purified ribosomes were collected from the resulting supernatant by centrifugation at lO5 o o o x g for 9° min.

CsCl density-gradient cenfri/ugation o/the puri/ied ribosomes Approx. I m g of the purified ribosomes was suspended in 3.2 ml of 5.3 M CsC1 solution (density 1.65) containing o.I M Tris and 0.03 M magnesium acetate (pH 7.2). A I-ml layer ot mineral oil was placed over the suspension and the centrifugation was carried out at 37 ooo rev./min for 36 h at 4 ° in a Spinco-L ultracentrifuge, SW 39 swinging bucket rotor. Fractions of one drop each were collected after piercing the bottom of the tube with a hyperdermic needle. The absorbancy at 260 m/~ of alternate fractions was measured and their content of cold TCA-precipitable radioactivity was assayed as follows: I ml each of I mg/ml crystalline bovine serum albumin and cold IO °/o TCA were added to each fraction and, after centrifugation in the cold, the precipitates were washed with cold 5 % TCA, dissolved in formic acid and planchetted.

Sucrose density-gradient centri/ugation o/ the crude extracts Crude extracts were prepared with o.005 M Tris containing o.oooi M magnesium acetate (pH 7.3), so that the ribosomes were mainly 5° S and 30 S (see ref. 6). After being freed of alumina, these extracts were treated with deoxyribonuclease (4/~g/ml). An aliquot of 0.4 ml of this extract was layered over 4.4 ml of linear sucrose gradient Biochim. Biophys. Acla, 61 (1962) 4 1 4 - 4 2 o

416

D. NAKADA, I. SMITH

(4 % to 20 % sucrose in 0.005 M Tris, o.oooi M magnesium acetate, pH 7-3) and centrifugation was carried out at 37 ooo rev./min for 75 min at 4 ° in a Spinco-L ultracentrifuge, SW 39 rotor. Fractions were collected, their absorbancy at 260 m/, was measured and their content of cold TCA-precipitable radioactivity was assayed. Colorimetric analysis o/ R N A and protein Aliquots of 4 ml of culture were treated with I ml of cold 25 % TCA. These samples were stored at 4 °, then centrifuged and the pellet was washed once with Cold 5 % TCA. The washed pellet was resuspended in 4 ml of 5 % TCA and heated for 3 ° rain at IOO°, then cooled and centrifuged. Aliquots of the supernatant were assayed for RNA by the orcinol method 7. The pellet was dissolved in I N NaOH and assayed for protein by the phenol method s . Chemicals 15NH4C1, 98.3 atom % ~5N, was obtained from Isomet Corp., Pallisades Park, New Jersey. "Iris (Sigma 121) was obtained from Sigma Chemical Company, St. Louis, Missouri. Optical-grade CsC1 was supplied by Maywood Chemical Works, Maywood, New Jersey. IS-laC]Adenine sulfate, specific activity 4.85 mC/mmole, was obtained from Nuclear Chicago Corporation, Chicago, Illinois, and L-II-14Clleucine, specific activity 5.15 mC/mmole, from New England Nuclear Corp., Boston, Massachusetts. Crystalline grade deoxyribonuclease was a product of Worthington Biochemical Corporation, Freehold, New Jersey. RESULTS AND DISCUSSION In the present experiments the separation of heavy and light ribosomes depends on their differential labelling with 15N and I'N, respectively. The difference in the density of these heavy and light ribosomes would not be expected to be as large as the difference found by BRENNERet al. 4 between 15N-13C-and 14N-12C-labelled ribosomes. Some control experiments were therefore performed. Radioactive l~N-labelled bacteria which had grown in a 15N-medium containing I14Cladenine were mixed with non-radioactive 14N-labelled bacteria in a ratio of I :50. Purified ribosomes were prepared from this mixture and subjected to centrifugation through a CsCIdensity gradient*. The results (Fig. I) showthat the radioactivity peak which identifies the heavy 15N-ribosomes is significantly displaced from the absorbancy peak which identifies the light taN-ribosomes. Another control was designed to determine the banding pattern in a case where new ribosomes have been made. 1~NLabelled cells were starved of uracil in a 14N-medium for 3° min; uracil was then added along with [8-"Cladenine and growth was allowed to proceed for 12 min during which time the turbidity of the culture increased by 12 %; thus, radioactive taN-ribosomes were made in the presence of preexisting 15N'-ribosomes. The results of the CsC] density-gradient analysis of the ribosomes of the culture are shown in Fig. 2. It * In the present CsCIdensity-gradient centrifugations on]yone distinct ribosomeband is seen while }3RENNERet al. 4 have observed two ribosome bands (Aand ]3bands, by their terminology). This apparent discrepancy can be explained by the fact that the ribosomes used in the present experiments were repeatedly purified before the final density-gradient centrifugation whereas in BRENNER et al.'s experiments relatively unpurified ribosomes were used. It has been reported recently that an unknown factor in the soluble fraction caused the separation of A and ]3 bands in the CsC1 density-gradient centrifugation 13. Biochim. Biophys. Acta, 61 (1962) 414-42o

4i 7

STABILITY OF RIBOSOMES DURING STARVATION

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Fig. i. D i s t r i b u t i o n of laN- a n d 14N-ribosomes in a CsC1 d e n s i t y g r a d i e n t . E. coli 63-86, a ura c i l less m u t a n t , g r o w n in a ~SN-medium c o n t a i n i n g I~4C]adenine w e re m i x e d w i t h a 5o-fold excess of t h e s a m e b a c t e r i a g r o w n in a ~4N-~2C-medium, a n d t h e r i b o s o m e s were p r e p a r e d . The p u r i f i e d rib o s o m e s were c e n t r i f u g e d a t 37 ooo r e v . / m i n for 36 h in 3.2 m l CsC1 ( d e n s i t y r.65) c o n t a i n i n g o. r M Tris, o.o 3 M m a g n e s i u m a c e t a t e (pH 7.2). © - O , a b s o r b a n c v a t 26o m/2; • - • , radioa c t i v i t y p r e c i p i t a b l e w i t h cold TCA. Fig. 2. D i s t r i b u t i o n of p r e - e x i s t i n g a n d n e w l y f o r m e d r i b o s o m e s in a CsC1 d e n s i t y g r a d i e n t . E. coli 63-86 was g r o w n in a l ~ N - m e d i u m a n d t r a n s f e r r e d t o a 14N-medium l a c k i n g ura c i l ; a f t e r 3 ° m in [14C]adenine t o g e t h e r w i t h u r a c i l w a s a d d e d a n d t h e b a c t e r i a l g r o w t h w a s a l l o w e d t o proc e e d for 12 rain. A t t h i s time, r i b o s o m e s were p r e p a r e d a n d puri fi e d; t h e p u r i f i e d r i b o s o m e s w e re cent r i f u g e d in CsC1 as d e s c r i b e d in t h e legend for Fig. i. O - O , a b s o r b a n c y a t 26o/zm; • • , r a d i o a c t i v i t y p r e c i p i t a b l e w i t h cold TCA.

can be seen that the radioactivity peak which identifies the newly formed light ribosomes is displaced towards a lighter density from the absorbancy peak which is due to the heavy preexisting ribosomes. The CsC1 density-gradient patterns of ribosomes prepared from lSN-grown bacteria which were starved of uracil in a 14N-medium for an initial 3o min then for 6o min in the presence of L-IIJ4Clleucine or [8-14Cladenine are illustrated in Figs. 3 and 4, respectively. During this 6o-min period of uracil starvation, no net increase in total protein and in RNA was observed but the radioactive precursors were incorporated into the TCA-precipitable fraction of the cells. Contrary to previous observations a on tile turnover of the structural protein of ribosomes, [14Clleucine was not incorporated into ribosomal protein (Fig. 3)- Radioactivity was found only at the meniscus where free protein would be expected to band; this may be due to the release of newly formed protein from the surface of tile ribosomes during the CsC1 density-gradient centrifugation. It is not likely that this protein was merely adsorbed to the ribosomes since tile ribosomes were thoroughly washed, a procedure which should remove any adsorbed protein"a0. Similar results were obtained when a methionine-less mutant of E. coli, KI2-4ooo, was grown in an ~SN-medium, starved of methionine in an t4N-medium for 3o min and then given L-[I-14Clleucine for 60 min. The CsC1 density-gradient pattern of the purified ribosomes showed TCA-precipitable radioactivity onlv at the meniscus. From Biochi~.. Hiophy,~

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Fig. 3- D i s t r i b u t i o n of ribosomes and n e w l y formed p r o t e i n in a CsC1 d e n s i t y gradient. E. coli 63-86 was g r o w n in a 15N-medium a n d transferred to a 14N-medium lacking uracil; after 3 ° min [14C]leucine was added and the uracil s t a r v a t i o n was continued for a f u r t h e r 6o min. At this time, ribosomes were p r e p a r e d a n d purified; the purified ribosomes were centrifuged as described in the legend for Fig. i. O - G , a b s o r b a n c y at 260 rap; • - • , radioactivity precipitable with cold TCA. Fig. 4. D i s t r i b u t i o n of ribosomes and newly formed lZNA in a CsC1 density gradient. E. coli 63-86 was g r o w n in a 15N-medium and transferred to a 14N-medium lacking uracil; after 3 ° min [laC ladenine was added and the uracil s t a r v a t i o n was continued for a f u r t h e r 60 min. At this time, ribosomes were p r e p a r e d and purified. The purified ribosomes were centrifuged as described in the legend for Fig. i. O - C ) , a b s o r b a n c y a t 26o m/z; • - O , radioactivity precipitable with cold TCA.

these results it can be concluded that new ribosomal protein is not being made under conditions of uracil or methionine starvation. The difference between the observations reported here and those of MANDELSTAM AND HALVORSON3 could be explained by the different condition of starvation used or by the method used to prepare the ribosomes; these authors determined the radioactivity of ribosomes that had been washed b y a few cycles of differential centrifugation. It is shown here that radioactive protein can be removed from washed ribosomes b y CsC1 density-gradient centrifugation. When the uracil starvation was carried out in the presence of [laC]adenine, the CsC1 density-gradient pattern of the purified ribosomes (Fig. 4) revealed that the radioactivity peak was directly in the center of the absorbancy peak which represents the preexisting 15N-labelled ribosomes. This indicates that new ribosomal RNA is not made under the condition of uracil starvation and agrees with the present observations on the stability of ribosomal protein. There are two possible explanations for the overlapping peaks of radioactivity and of absorbancy: either, since the ribosomes were prepared and analyzed in the presence of a high concentration of magnesium, the "messenger RNA ''1~ could be attached to the ribosomes and thus be responsible for the observed readioactivity peak; or [14C]adenine could have been incorporated into preexisting ribosomal RNA due to partial turnover. To distinguish between these possibilities, experiments using sucrose densitygradient centrifugation in a low concentration of magnesium to allow the separation of messenger RNA was carried out. The messenger RNA of uracil-starved cells which had been exposed to [laCladenine for 3 min was highly labelled; little incorporation Biochim. Biophys. ,4eta, 61 (1962) 414-42o

419

STABILITY OF RIBOSOMES DURING STARVATION

was observed in the soluble fraction (Fig. 5a). When the exposure to [t4Cladenine was continued for 6o min under uracil starvation, most of the radioactivity was found in the soluble fraction though the messenger-RNA fraction still had discernible activity (Fig. 5b). In both experiments there was little, if any, incorporation into ribosomal

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RNA; this is in contrast to the extensive incorporation of radioactivity into ribosomes (both 5o S and 3o S) when a radioactive purine or pyrimidine is added during growth 6,t2. The present experiments show high uptake into both messenger and soluble RNA but not into ribosomal RNA and strongly indicate the stability of ribosomes during uracil starvation. The conclusion which m a y be drawn from these results is that neither synthesis of new ribosomes nor turnover of ribosomal RNA and protein occurs during starvation for RNA or protein precursors.

ACKNOWLEDGEMENTS This work was aided in part by grants from the National Science Foundation and tile Public Health Service administered by Professor F. ,]. RYAN, Department of Zoology, Columbia University, New York, New York, and by a grant from the National Institutes of Health, No. CY-52IO, administered by Professor B. MAGASANIK, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts. The authors wish to express their thanks to Professors RYAN and MAGASANIK for their support and criticism of this work. Biochim. Biophys. Acta, 61 (1962) 414 42o

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REFERENCES I. DAVERN AND M. MESELSON, J. Mol. Biol., 2 (196o) 153. GOLDSTEIN AND B. J. BROWN, Biochim. Biophys. Acta, 44 (196o) 491. MANDELSTAMAND H. HALVORSON, Biochim. Biophys. Acta, 4 ° (196o) 43. BRENNER, F. JACOB AND M. MESELSON, Nature, 19o (1961) 576. NAKADA, Biochim. Biophys. Acta, 55 (1962) 505 . GROS, W. GILBERT, H. HIATT, C. KURLAND, R. W. RISEBROUGH AND J. D. WATSON, Nature, 19o (1961) 581. 2 W. MEJBAUM, Z. physiol. Chem. Hoppe Seyler's, 258 (1939) 117. O. H. LOWRY, N. J. ROSENBROUGH, A. L. TORR AND R. T. RANDALL, J. Biol. Chem., 193 (I95 I) 265. 9 D. B. COWIE, S. SPIEGELMAN, R. B. ROBERTS AND J. D. DUERKSEN, Proc. Natl. Acad. Sci. U.S., 47 (1961) 114 . 10 H. K. KIHARA, A. S. HU AND I-I. O. HALVORSON, Proc. Natl. Acad. Sci. U.S., 47 (1961) 489 . n F. JACOB AND J. MONOD, J. Mol. Biol., 3 (1961) 318. la F. GROS, W. GILBERT, H. H. HIATT, G. ATTARDI, P. F. SPAHR AND J. D. WATSON, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) i i i . 1~ M. MESELSON, p e r s o n a l c o m m u n i c a t i o n . 1 2 3 * 6

C. A. j. S. D. F.

Biochim. Biophys. Acta, 61 (1962) 414-42o