Biogenesis of 30-S subunits during recovery from inhibition of protein synthesis

352

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 97157

BIOGENESIS OF 3o-S SUBUNITS DURING RECOVERY FROM I N H I B I T I O N OF P R O T E I N SYNTHESIS

F. C. D A V I S a AND B. H. S E L L S b' *

aDepartment o/ Zoology, University o[ Florida, Gainesville, Fla. and bLaboratory o/Biochemistry, St. Jude Children's Research Hospital, Memphis, Tenn. 38zoz (U.S.A.) (Received October I8th, 1971)

SUMMARY

The rate of synthesis of ribosomal proteins was determined early during recovery from (i) puromycin treatment, (ii) chloramphenicol treatment, and Off) from methionine starvation. Patterns of ribosomal protein synthesis were similar, but not identical during recovery of the cells from the different treatments. Early during recovery production of several proteins is significantly increased while synthesis of another group is significantly reduced. Normal patterns of ribosomal protein synthesis were observed after 60-65 rain of recovery from chloramphenicol treatment. At 20-25 rain following recovery the proteins whose synthesis were elevated early were decreased; on the other hand, synthesis of those proteins which were depressed early during recovery were elevated. The results are interpreted to suggest that there is unbalanced accumulation of information for synthesis of 3o-S ribosomal protein.

INTRODUCTION

Previous studies have revealed that during recovery from chloramphenicol or puromycin treatment Escherichia coli preferential synthesize ribosomal protein over soluble protein 1'4. Similar findings have been reported for recovery from methionine starvation in E. coli ( R C rel) strain 2,3. A more recent examination of 5o-S ribosomal proteins has indicated that during recovery from chloramphenicol, puromycin treatment, or methionine starvation not all the structural proteins are synthesized at the same rate 3-5. Further study has also revealed that the time at which r-protein synthesis is measured during recovery influences the results obtained. Proteins synthesized to the greatest extent early during recovery from chloramphenicol treatment were synthesized to the lowest extent during recovery4. The present communication reports the observations made on a study of the formation of 3o-S ribosomal protein during recovery from chloramphenicol treatment. These results are compared with data obtained from studies during recovery from puromycin treatment and from methionine starvation. To w h o m correspondence should be addressed.

Biochim. Biophys. Acta, 262 (1972) 352-359

BIOGENESIS OF 30-S SUBUNITS

353

MATERIALS AND METHODS

Chemicals L-[I-14C]Leucine (3o mC/mmole) and L- E4,5-3H~leucine (5 C/mmole) were obtained from New England Nuclear Corporation. Chloramphenicol was donated b y Parke Davis and Co. Puromycin was purchased from Nutritional Biochemicals Corporation. All other chemicals were reagent grade and readily obtainable from commercial suppliers.

Bacteria and cultural conditions The bacteria, E. coli KT11 (RC rel) methionine and arginine-requiring relaxed mutant, was used throughout this study. The conditions used for growth have been described 4. When bacterial cells attained an As~5 nm of O.I, L-E4,5-aHlleucine (1. 5 mC/ ml) at a final concentration of 15 p g / m l was added. Chloramphenicol or puromycin was added to the cell suspension when the absorbance reached 0.8 (approx. 7 " l°S cells/ml). In the case when methionine starvation was employed to arrest protein synthesis the cells were sedimented and resuspended in fresh warmed (37 °) medium lacking methionine. In each case the incubation was continued for 30 min and then terminated b y pouring the cells over crushed ice (--20°). Cells were collected b y sedimentation at 0-4 ° and resuspended in pre-warmed media. L-~I-14C]Leucine (0.0 4 #C and o.o5 mg/ml) unless otherwise indicated was added at intervals as noted in the text.

Preparation o/3o-S Subunits Bacteria collected by centrifugation were washed with cold buffer containing lO _2 M Tris-HC1, 6 . lO -3 M mercaptoethanol and 3" IO-2 M NH4C1 having a p H of 7.4 e and supplemented with IO-z M magnesium acetate. The cells then were resuspended in 15 ml Tris-mercaptoethanol-NHaC1 (IO-2 M Mg 2+) and frozen a t - - 2 o ° overnight. All subsequent steps were performed at 0-4 °. The cells were broken in a French pressure cell at 6oo0-8ooo lb/inch 2 and the total ribosome fraction isolated as previously described 4. The 3o-S subunits were obtained and the ribosomal proteins extracted and fractionated as outlined in an earlier communication 7. RESULTS

Synthesis o~ ribosomal protein during intervals o/ recovery /rom chloramphenicol The following expeliment was designed to determine the pattern of ribosomal protein synthesis during valious 5-min intervals of recovery from chloramphenicol. Cells prelabeled with E3HJleucine were treated with chloramphenicol for 30 min and transferred to medium free of chloramphenicol. During recovery cells were incubated during the following intervals: 0-5 rain, 20-25 min and 60-65 rain with E14C]leucine, (o.o7/~C and o.oo382#mole/ml ). At the end of each labeling interval a Ioo-fold excess of unlabeled leucine was added and incubation continued for 60 rain for the o-5-min labeling period and 3o rain for the 20-25- and 6o-65-min labeling period. These incubation times were previously shown to be sufficiently long to permit all labeled ribosomal protein to be incorporated into complete ribosomes 4. At the end of the incubation peliod cells were poured over crushed ice and collected. The patterns of labeling for each interval are shown in Fig. I and Table I.

Biochim. Biophys. Acta, 262 (1972) 352-359

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50 40 50 60 70 80 SLICE NUMBER Fig. I. P o l y a c r y l a m i d e gel electrophoresis of 3o-S r i b o s o m a l proteins. Cells were prelabeled w i t h [3H]leucine for 3 g e n e r a t i o n s a n d labeled w i t h p*C]leucine a t (a) o - 5 - m i n , (b) 20-25 m i n a n d (c) 6 o - 6 5 - m i n i n t e r v a l s d u r i n g r e c o v e r y f r o m c h l o r a m p h e n i c o l . I n c u b a t i o n s were c o n t i n u e d in t h e p r e s e n c e of a I5o-fold excess of labeled lencine for a n a d d i t i o n a l 6o min. 3o-S s u b u n i t s were isolated a n d profiles of r a d i o a c t i v e r i b o s o m a l p r o t e i n s o b t a i n e d as described in t h e t e x t . - - , [SHJl e u c i n e ; - - - , [liC]leucine.

BIOGENESIS OF TABLE

I

ISOTOPE

CONTENT

FROM

3o-S

OF

SUBUNITS

3O-8

CHLORAMPHENICOL

RIBOSOMAL

355

PROTEINS

LABELED

AT V A R I O U S

INTERVALS

OF RECOVERY

TREATMENT

T h e d a t a p r e s e n t e d in t h i s t a b l e r e p r e s e n t s i n f o r m a t i o n d e r i v e d f r o m Fig. i. To l o c a t e t h e slice c o n t a i n i n g each b a n d , t h e d i s t a n c e o b t a i n e d f r o m a p h o t o g r a p h of e a c h gel a n d t h e n u m b e r of gel slices s e p a r a t i n g B a n d s 6 a n d 12 were d e t e r m i n e d . F r o m t h e s e v a l u e s a r a t i o ( n u m b e r of gel s l i c e s / d i s t a n c e ) were c a l c u l a t e d . U s i n g a s i m p l e p r o p o r t i o n t h e v a l u e s c a l c u l a t e d f r o m t h i s r a t i o c a n b e m u l t i p l i e d b y t h e d i s t a n c e of a g i v e n b a n d f r o m B a n d 12 t o y i e l d t h e slice n u m b e r s i n w h i c h t h e b a n d w a s located. T h e I*C/3H r a t i o for each b a n d w a s c a l c u l a t e d a n d t h e v a l u e s n o r m a l i z e d as p r e v i o u s l y describedL

Band No.

Normalized ratios 0-5 rain

20-25 rain

60-65 rain

I

0.89

--

0.86

2 3 4 5 6 7

0.85 0.85 1.91 1.28 0.94 1.34

1.12 1.17 o.68 I.O 4 I. I I 0.85

i.oo I.OO I.O2 o.95 I.O 4 0.99

8

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o.79 0.89 o.87 0.92 I.OI 1.48 1.21 0.75 0.66 0.64 0.70

1.39 1.2o 1.23 1.o8 -0,98 o,98 o,73 o.77 o.82 0.84

1.o3 1.o6 1.o 4 0.99 -I .o2 1.o6 0.9o 1.o2 1.o2 1.o7

9 IO II 12 13 14 15 16 17 18 19

The patterns of ribosomal protein synthesis varied during intervals of recovery from chloramphenicol treatment. During the first 5 min the 14C/3H ratios were highest for Bands 4,5,7,i4 and 15. During the 2o-25-min interval the 14C/3H of these bands had decreased while the ratios of Bands 2, 3, 9, IO and I I which previously had been low were elevated. By the interval 60-65 rain the cells had recovered and a normal pattern of ribosomal protein synthesis was obtained.

Assembly o/ proteins labeled early during recovery/rom (i) puromvcin treatment and (ii) methionine starvation To establish whether a similar pattern of ribosomal synthesis occurs early during recovery from puromycin treatment or from methionine starvation as was observed during recovery from chloramphenicol treatment the following experiments were performed. Bacterial cells prelabeled with ESHJleucine were either treated for 4 ° min with puromycin or stalved 30 min for methionine. The cells then were allowed to recover from the above treatments b y removal of the drug or addition of methionine. Early during recovery E14C]leucine was added for a short interval (5 min for puromycin treated cells and 2 rain for methionine-starved cells) followed b y a period during which the cells were incubated with a ioo-fold excess of unlabeled leucine for 60 rain. The cells were then harvested, 3o-S subunits isolated and ribosomal Biochim. Biophys, Acta, 262 (i972) 352-359

356

F . C . DAVIS, B. H. SELLS

proteins extracted and processed on polyacrylamide gels. The results of these experiments described in Table II, Fig. 2 compare the rates of synthesis of 3o-S ribosomal protein during recovery in the 3 systems and reveals a marked similarity in the 3o-S ribosomal proteins synthesized.

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Fig. 2. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of 3o-S r i b o s o m a l p r o t e i n s f r o m cells p r e l a b e l e d w i t h [SH]leucine a n d p r e l a b e l e d w i t h [liC]leucine e a r l y d u r i n g r e c o v e r y (a) from p u r o m y c i n t r e a t m e n t a n d (b) f r o m m e t h i o n i n e s t a r v a t i o n . See t e x t for details. , [3H]leucine, - - -, [14C]leucine.

Biochim. Biophys. Acta, 262 (1972) 352-359

357

BIOGENESIS OF 3 0 - S SUBUNITS T A B L E II ISOTOPE OR

CONTENT

PUROMYCIN

OF

3o-S

TREATMENT

PROTEINS AND

LABELED

FROM

EARLY

METHIONINE

DURING

RECOVERY

FROM

CHLORAMPHENICOL

STARVATION

T h e d a t a p r e s e n t e d in t h i s t a b l e r e p r e s e n t s d a t a o b t a i n e d f r o m Figs. I a n d 2.

Band No.

Normalized ratios Chloramphenicol

Puromycin

Methionine

1 2 3 4 5 6 7

0.89 0.85 0-85 1.92 1.28 o.94 1.34

I.I2 0.94 0.85 1.6I 1.44 o.90 1-53

I.I 7 0.98 0.90 I.II 1.16 o.86 1-34

8

--

--

--

o-79 o.89 o.87 0.92 i.oi 1.48 1.21 0.75 0.66 0.64 0.70

0.86 i.oi 1.o 3 o.77 o.76 I.OO 0.98 0.82 o.73 0.79 --

0.92 1.19 1.16 o.91 0.92 1.o 5 o.91 -o.76 o.81 0.80

9 Io Ii i2 13 14 15 16 17 18 19

DISCUSSION

Unequal production of individual 3o-S ribosomal proteins occurs early during recovery of E. cull from inhibition of protein synthesis by either puromycin or chloramphenicol treatment or methionine starvation. The patterns of ribosomal protein synthesis are similar but not identical during recovery of the cells from the different treatment. The synthesis of proteins in Bands 4, 5 and 7 is significantly elevated during recovery in each case while production of proteins of proteins in Bands I6, 17, I8 and 19 is lowered significantly. Synthesis of proteins in other bands shows consistent trends during recovery in all cases but the deviation from the average is not significant when compared with normal variation observed with ribosomal proteins labeled during exponential growthL The labeling pattern obtained in several bands is determined by the type of previous inhibition of protein synthesis. For example the pattern of synthesis of protein in Bands IO and IX during recovery from methionine starvation, in Bands x2 and I3 during recovery from puromycin inhibition and in Bands 14 and 15 during recovery from chloramphenicol inhibition is dependent upon the treatment. Analogous results have been observed in synthesis of 5o-S ribosomal proteins (3-5), however, greater similarity has been noted in the patterns of 5o-S ribosomal protein production. These studies demonstrate that during recovery from inhibited protein synthesis an unbalanced production of the various 3o-S ribosomal proteins occurs and that their pattern of synthesis is similar regardless of the procedure used to inhibit protein synthesis. Biochim. Biophys. Acta, 262 (1972) 352-359

358

F . C . DAVIS, B. H. SELLS

The data obtained in these studies suggest that information for the formation of ribosomal proteins accumulated during inhibition of protein synthesis and was expressed during recovery. In support of this contention SELLS AND SAYLER$ have observed that in the absence of RNA synthesis certain ribosomal proteins are preferentially produced in E . coli recovering from treatment with chloramphenicol. Similar results have been reported by HANEY AND NAKADA9 in Bacillus subtilis recovering from puromycin treatment and in the presence of actinomycin. These results support the belief that there is unbalanced accumulation of information for synthesis of 3o-S ribosomal protein; however, the results do not eliminate the possibility that the control of unbalanced synthesis is at the translational level. The pattern of ribosomal protein synthesis observed early after removal of chloramphenicol is not maintained through the entire recovery period. The proteins which are preferentially synthesized during the first 5 min of recovery from chloramphenicol treatment are synthesized to a lesser extent during 20-25 rain of recovery (Table I). Conversely, the proteins which are synthesized to a lesser extent during the first 5 rain are preferentially synthesized during 20-25 min of recovery. Similar results were observed with the pattern of synthesis of 5o-S ribosomal proteins during recovery from chloramphenicol treatment 4. The proteins in Bands 16, 17, 18 and 19 are an exception to this general trend and are synthesized to a lesser extent than the average of all ribosomal proteins during the intervals of recovery examined. By the 6o-65-min interval of recovery the cells have fully recovered and a normal pattern of synthesis of 3o-S ribosomal proteins is observed. The pattern of synthesis and assembly of proteins in Bands 16, 17 , 18 and 19 is unlike other 3o-S and 5o-S ribosomal proteins. The proteins in Bands 17, 18 and 19 were previously shown to be the 3o-S ribosomal proteins most rapidly labeled during the period following a short pulse with a labeled amino acid in exponentially growing E . coli ~. Early during recovery of cells from protein synthesis inhibition the synthesis of proteins in Bands 16, 17, 18 and 19 is less than the average of all ribosomal proteins. These results are unlike those observed for the synthesis of 5o-S ribosomal proteins which revealed that the proteins synthesized and assembled most rapidly in exponentially growing cells1° were also preferentially synthesized during recovery from inhibition of protein synthesis (3-5). The proteins in Bands 16, 17, 18 and 19 are not preferentially synthesized during either the interval 0-5 or 20-25 min after removal of chloramphenicol (Table I) unlike essentially all other 3o-S and 5o-S ribosomal proteins 4. This depressed synthesis of Band 16, 17, 18 and 19 during recovery period from inhibition of protein synthesis is similar to that of the bulk of the nonribosomal proteins 4. The variation in the pattern of synthesis and assembly of proteins in Bands 16, 17, 18 and 19 from other ribosomal proteins suggest that their synthesis is under a separate control from the remainder of the 3o-S ribosomal proteins. Comparison of our data with that of TRAUT et ul. ~ suggests that the proteins in Bands 16, 17, 18 and 19 may be among those present in less than one copy per subunit. KURLAND el al. 12 proposed that the proteins present in less than one copy per subunit exchange during the protein synthetic cycle. These observations indicate that certain proteins are not a permanent portion of the 3o-S subunit structure and are consistent with the suggestion that the synthesis of the proteins in Bands I6,I 7, 18 and 19 is under a separate control from the remainder of the 3o-S ribosomal proteins. Biochim. Biophys. Acta, 262 (1972) 352-359

BIOGENESIS OF

3o-S SUBUNITS

359

ACKNOWLEDGMENTS

These studies were supported by the U.S. Public Health Service Grant AM07375 and the Damon Runyon Memorial Fund DRG-734. F.C.D. was a Research Trainee of the National Cancer Institute (ToI-CA-o5176). The excellent technical assistance of Mrs. Jane Sayler is gratefully acknowledged. REFERENCES H. SELLS, Biochim. Biophys. Acta, 80 (I964) 230. NAKADA, I. A. C. ANDERSON AND B. 1V[AGASE~IICK, J. Mol. Biol., 9 (I964) 472. C. DAVIS AND B. H. SELLS, J. Mol. Biol., 49 (I97 °) 527. C. D A v l s AND B. H. SELLS, J. Mol. Biol., 39 (1969) 503 • H. SELLS AND J. SAYLER, Biochim. Biophys. Acta, 232 (1971) 736. TRAUB, M. NOMURA AND L. TU, J. Mol. Biol., 19 (1966) 215. C. DAVIS AND B. I"I. SELLS, Biochim. Biophys. Acta, 232 (1971) 379I-[. SELLS AND J. SAYLER, Biochim. Biophys. Acta, 232 (1971) 421. E. HANEY AND D. NAKADA, Biochim. Biophys. Acta, 213 (197 o) 529. IO B . I-I. SELLS AND F. C. DAVIS, J. Mol. Biol., 47 (197 o) 155. i i R. R. TRAUT, H. DELIUS, C. AHMAD-ZADISH, T. A. BICKLE, P. PEARSON AND A. TlSSlERES, Cold Spring Harbor Syrnp. Quant. Biol., 34 (1969) 25. 12 C. G. KURLAND, P. VOYNOW, S. J. S. HARDY, L. RANDAL AND L. LUTTER, Cold Spring Harbor Syrup. Quant. Biol., 34 (1969) I7. I 2 3 4 5 6 7 8 9

B. D. F. F. B. P. F. B. C.

Biochim. Biophys. Acta, 262 (1972) 352-359