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Control of stable-RNA synthesis and maturation of 5-S RNA in Salmonella typhimurium RCstr and RCrel
314
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96762
CONTROL OF STABLE-RNA S Y N T H E S I S AND M A T U R A T I O N OF 5-S RNA IN S A L M O N E L L A T Y P H I M U R I U M RC str AND RC tel
H. A. RAUI~ AND M. G R U B E R
Biochemisch Laboralorium, The University, Groningen (The Netherlands) (Received S e p t e m b e r I Ith, 197 o)
SUMMARY
I. Synthesis of stable RNA in Salmonella typhimurium RC str and RC re1 during amino acid starvation was studied with the aid of methylated albumin kieselguhr chromatography and polyacrylamide gel electrophoresis. 2. Synthesis of all classes of stable RNA was coordinately controlled by the RC gene under these conditions. The effect of the RC gene therefore appears to be the same in Escherichia coli and S. typhimurium. 3. Amino acid starvation did not result in the accumulation of an abnormal form of 5-S RNA, as in E. coli. However, addition of chloramphenicol, to either starved or exponentially growing cells, did result in the accumulation of such an abnormal form, with properties very similar to those of the E. coli 5-S RNA precursor. 4. Experiments with various low doses of chloramphenicol, indicate that maturation of 5-S RNA is dependent on a minimum rate of protein synthesis, which, under our conditions, lies between 4 ° °/o and 60 ~o of the rate shown by exponentially growing cells.
INTRODUCTION
Regulation of synthesis of stable RNA in bacteria has been widely studied since the discovery of the RC tel mutants of Escherichia coli. In contrast to the wild type (RCstr), the rate of RNA synthesis in these mutants is hardly affected b y amino acid starvation. (For a review see ref. I.) From the work of different groups 2,3, it appears that, in this organism, synthesis of t R N A and high moleculal weight RNA is coordinately controlled b y amino acid starvation (see, however, ref. 4). With respect to the control of 5-S RNA synthesis there is less information. Levallorphan inhibits synthesis of 5-S RNA and high molecular weight rRNA to the same extent, while t R N A synthesis is inhibited to a lesser extent 5. Starved E. coli RC tel cells seem to synthesize a normal amount of a precursor e as do wild type cells, treated with high doses of chloramphenicol (see refs. 7, 8). The same precursor has been found in starved RC str cells4. The effect of amino acid starvation on RNA synthesis in organisms other than E. coli has hardly been studied, probably because the only other RC re1 mutants known are those of Salmonella typhimurium. Biochim. Biophys. Acta, 232 (1971) 314-323
RNA SYNTHESIS IN S. typhimurium
315
We studied the synthesis of stable RNA, including 5-S RNA, during amino acid starvation, in two S. typhimurium strains, which, as far as is known, are isogenic except for the RC gene. In the course of these studies, we encountered an abnormal form of 5-S RNA, which showed properties similar to those of the above-mentioned E. coli 5-S RNA precursor.
MATERIALS AND METHODS
Bacterial strains Two Salmonella typhimurium strains with different control of RNA synthesis (i.e. RC str and RC re1) were obtained from Dr. A. R6rsch (Medisch Biologisch Laboratorture R.V.O.-T.N.O., Rijswijk, Nethellands). These strains were originally isolated b y Dr. A. Eisenstark and aie, as far as is known, isogenic except for the RC gene, since the relaxed strain was obtained b y bringing the RC tel gene from E. colt into wild type (RC str) S. typhimurium.
Growth conditions Cells were grown at 37 ° in flasks on a gyrotory shaker in the minimal medium described b y SCHAECHTER et al. 9, supplemented with o.2 % glycerol; 20 ffg/ml cytosine was added to reduce incorporation of uracil into DNA 1°. In measurement of RNA and protein synthesis, b y incorporation of labeled uracil or leucine, 20 ffg/ml unlabeled uracil and L-leucine were also included in the medium. Growth was followed spectropho tometrically. An overnight culture of the desired strain in minimal medium plus 0.2 % glycerol was diluted to an A450 nm of 0.03 ( I - c m path length) into fresh prewarmed medium, containing the necessary supplements. When an A45onm of 0. 3 was reached, the culture was divided into subcultures for the desiled treatment. Carrier cells were obtained b y harvesting an exponentially growing culture in the late log phase. The cells were washed once with cold Tris-MgCl 2 buffer (o.oi M Tris (pH 7.5)-O.Ol M MgCI~) and stored in aliquots of 0.6 g at --20 °.
Amino acid starvation and chloramphenicol treatment The subcultures (usually 50 ml) were subjected to amino acid starvation b y adding 20 ffg/ml 5-DL-methyltryptophan (Sigma Chem. Co., St. Louis, Mo., U.S.A.) which results in a lack of t r y p t o p h a n in the cells n. RNA was labeled b y addition of [5-3Hluracil 30 rain after addition of 5-methyltryptophan. Chloramphenicol treatment in the presence of 5-methyltryptophan, was carried out b y adding 2.5 ffg/ml chloramphenicol to a culture 30 min after addition of 5-methyltryptophan. [5-~H]Uracil was added al the same time. Chloramphenicol treatment in the absence of 5-methyltryptophan was carried out b y adding chloramphenicol to a subculture with an A45o nm of 0. 3. Incubation was continued for 30 min and [5-3Hluracil was added. In all cases incubation was stopped 60 rain after addition of the label and cells were stored at --20 ° after washing with cold Tris-MgCl 2 buffer. The amount of [3Hluracil and the concentrations of chloramphenicol used are mentioned in the legends. Biochim. Biophys. Acta, 232 (1971) 314-323
316
H . A . RAU]~, M. GRUBER
Control RNA was obtained b y labeling an exponentially growing culture of the desired strain with o.4/~C/ml [2-14C]uracil for 9 ° min from the time an A45onm of 0.3 was reached. In some cases, the label of the treated and control cells was reversed.
Measurement o] RNA and protein synthesis o.I #C/ml of either ~2-14C~uracil or L-[14Clleucine was added at time o together with 20/zg/ml 5-methyltryptophan to a subculture with an A45o nm ot 0.3. Duplicate samples of I.O ml were taken at times indicated. Samples were mixed immediately with I.O ml of ice-cold IO % trichloroacetic acid and left overnight at 4 °. Samples from a control culture were obtained at the same time. The samples were processed for counting in the usual way by filtering and washing on membrane filters (Sartorius Membranfiltergesellschaft G6ttingen, W. Germany; MF5o). Filters were counted in a Nuclear Chicago Mark I liquid scintillation counter. All values were corrected for blanks taken at time o.
RNA extraction Labeled cells from 50 ml of treated culture were mixed with labeled cells flora 50 ml of control culture and 0.6 g of carrier cells in IO ml of cold Tris-MgC12 buffer containing I.O mg/ml egg-white lysozyme (Armour Pharmaceutical Co., Eastbourne, England) and o.oi mg/ml deoxyribonuclease (Nutritional Biochem. Co. Cleveland, Ohio, U.S.A.). The suspension was frozen in liquid nitrogen and thawed at room temperature. Ariel thawing, th~ suspension was incubated for io rain in an ice bath and then sonicated in an MSE ultrasonic disintegrator for I min at 18 ooo20 ooo cycles/sec while cooled in ice. After a further incubation for 5 rain in an ice bath, RNA was extracted with 80 % (v/v) of redistilled phenol in Tris-MgC12 buffer in the presence of 0.5 % sodium dodecyl sulfate, as described ~2. The RNA was dissolved in 5.0 ml of the starting buffer for methylated albumin kieselguhr chromatography and the concentration of the solution was determined by measuring the A260 nm. The Eo~.~O~oat 260 nm was taken as 20. The recovery of RNA, as determined by measurement of radioactivity, was between 85 and 95 %.
Methylated albumin kieselguhr chromatography and preparation of low molecular weight RNA Methylated albumin kieselguhr chromatography was carried out at 35 ° as described earlier lz. Radioactivity was measured b y mixing 0.5 ml of each fraction with IO ml of scintillation fluid (300 ml dioxan, 300 ml methylcellosolve, 33 ° ml toluene, 80 g naphtalene, 4 g PPO) and counting in a liquid scintillation counter. Low moleculor weight RNA for gel electrophoresis was prepared b y adsorbing 5-6 mg of total RNA on a methylated albumin kieselguhr column in the usual way. The column was eluted with 0.6 M NaC1 in phosphate buffer. The whole procedure was carried out at room temperature to prevent denaturation of 5-S RN & which takes place only at elevated temperatures. The fluid containing A 2~0nm absorbing material was collected in a test tube cooled in ice. This solution (20--25 ml) was dialysed against 50 vol. of distilled water at 4 ° for 4 h with one change after 2 h. The residue, obtained upon lyophilization, was dissolved in the buffer used for electrophoresis, without sodium dodecyl sulfate but containing io % sucrose. The concentration of this solution, determined a s A260 nm, was adjusted to 2 mg/ml. Biochim. Biophys. Acta, 232 (1971) 314-323
RNA SYNTHESIS IN S. typhimurium
317
Polyacrylamide gel electrophoresis Electrophoresis was carried out on gels containing IO % (w/v) acrylamide (British Drug Houses, Poole, England; recrystallized from chloroform) according to the method of WEINBERG et al. z3. However, pH was kept at 7.8 and crosslinker concentration was doubled, which resulted in a slightly better resolution. Electrophoresis time was 4 h at 60 V and 32 mA using 8 gels of 9 cm length. On each gel 20/zl of a solution of low molecular weight RNA was layered. After electrophoresis, the gels were cut into I-mm slices which were hydrolysed overnight in 0.5 ml of concentrated ammonia. Ammonia was removed and radioactivity was determined by adding IO ml of scintillation fluid and counting in a liquid scintillation counter. Radiochemicals [2J4C]Uracil (specific activity 52 mC/mmole), [5-3Hluracil (specific activity 23.3 mC/mmole) and L-[14C]leucine (specific activity 311 mC/mmole, uniformly labeled), were obtained from The Radiochemical Centre, Amersham, England. RESULTS
Control o/ stable-RNA synthesis As shown in Fig. I the two strains of S. typhimurium show a behaviour characteristic of an RC s~ and RCre] genotype respectively, under conditions of amino acid ~,r M
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Fig. i. Effect of amino acid s t a r v a t i o n on R N A and protein synthesis in S. typhimurium I~Catr a n d RC tel. To an exponentially growing culture of each strain, s u p p l e m e n t e d with uracil a n d leucine, o. I / z C / m l of either [z4C]uracil or [z4C]leucine was added, t o g e t h e r w i t h 2 o / , g / m l 5-methylt r y p t o p h a n . Samples were t a k e n at t h e times indicated, filtered and w a s h e d on m e m b r a n e filters, and counted in a liquid scintillation counter. I n c o r p o r a t i o n b y a control culture was determined in the same way. Panels A and ]3: R C str, Panels C and D: RC re]. O , 5 - m e t h y l t r y p t o p h a n treated; 0 , control. Fig. 2. F r a c t i o n a t i o n of double-labeled total R N A from a m i x t u r e of an a m i n o acid s t a r v e d a n d a control culture of S. typhimurium RC str, b y m e t h y l a t e d a l b u m i n kieselguhr c h r o m a t o g r a p h y . [3H]Uracil (8.0 #C/ml) was added to a culture of the RC str strain 3 ° min after t h e addition of 5 - m e t h y l t r y p t o p h a n . Cells were h a r v e s t e d 60 m i n later, mixed w i t h carrier cells, and [14CJuracillabeled control cells, a n d extracted w i t h phenol. I~NA was c h r o m a t o g r a p h e d on a m e t h y l at a l b u m i n kieselguhr c o l u m n (2.1 cm × 12 cm) a t 35 °.
Biochim. Biophys. Act(*, 232 (197I) 314-323
318
H . A . RAUE, M. GRUBER
starvation. Protein synthesis is inhibited to about the same extent in both strains. RNA synthesis in the RC str cells is depressed to about 20 %; in the RC rd cells no reduction in RNA synthesis can be detected up to 9 ° min after addition of 5-methyltryptophan. The residual protein synthesis is rather high in comparison to t h a t observed in auxotrophic strains during amino acid starvation. Further reduction, however, by increasing the concentration of 5-methyltryptophan, was impossible. Therefore, a concentration of 20#g/ml was routinely used. To study the synthesis of different species of RNA during amino acid starvation, chromatography on methylated albumin kieselguhr columns was used for a rough separation. Fig. 2 shows a typical chromatogram of double-labeled total RNA from amino acid starved and control RC str cells. A portion of the 5-S RNA is denatured on the column under the conditions used, hence this RNA is eluted in two peaks ~2. The labeling pattern of RNA isolated from a mixture of starved and control RC r~l cells was essentially the same, except for differences in the 23-S peak which are now under investigation. Since the recovery of RNA during isolation and chlomatography in most cases was better than 9 ° °/o, the labeling pattern allows us to conclude, that synthesis of t R N A and high molecular weight rRNA in S. typhimurium is coordinately controlled b y amino acid starvation. The same conclusion probably holds true for synthesis of 5-S RNA, but it is difficult to infer this unambiguously from the methylated albumin kieselguhr chromatogram, because of the incomplete resolution of the 5-S peaks. Therefore, we used polyacrylamide gel electrophoresis, which allows an almost complete separation of 5-S RNA and tRNA.
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Fig. 3. Synthesis of 5-S R N A b y S. typhimurium :RCstr and RC re1 during amino acid starvation. Low molecular weight R N A was p r e p a r e d from a m i x t u r e of [DH]uracil-labeled starved, and [14C]uracil-labeled control cells of either strain. I n the case of the RC str strain 8.0/~C/ml ~eH]uracil was used, in t h a t of the RC tel strain 4.o #C/ml. The R N A was electrophoresed on IO % (w/v) polya6rylamide gels. Gels were sliced, hydrolysed, and counted in a liquid scintillation counter. Panel A: RC str. Panel B: RC tel. I n the top of each panel the SH/x4c ratio is shown. Fig. 4- Synthesis of 5-S R N A in S. typhimurium RC tel, t r e a t e d with chloramphenicol in t h e presence of 5 - m e t h y l t r y p t o p h a n . Chloramphenicol (2. 5/~g/ml) was added to a culture of the RC rel strain, together with 0. 4 #C/ml [14C]uracil, 3 ° min after the addition of 5 - m e t h y l t r y t o p h a n . Cells were harvested 60 rain later, mixed with carrier cells, and control cells labeled with 4.0 I,C/ ml ESH]uracil and extracted with phenol. A p o r t i o n of the R N A obtained, was used for methylated a l b u m i n kieselguhr c h r o m a t o g r a p h y . L o w molecular weight RlqA was p r e p a r e d from the other portion, and electrophoresed on io % (w]v) polyacrylamide gels. Panel A: m e t h y l a t e d a l b u m i n kieselguhr c h r o m a t o g r a m ; only t h e first p a r t of the labeling p a t t e r n is shown. Panel B: electrophoretic p a t t e r n .
Biochim. Biophys. Acta, 232 (1971) 314-323
RNA SYNTHESIS IN S. typhimurium
319
Fig. 3 shows the electrophoretic pattern of low-molecular weight RNA: isolated from a mixture of starved and control cells of either strain. It is clear, that in either case, the ratio of 5-S RNA to tRNA is the same in starved and in exponentially growing cells, which indicates that synthesis of both species of RNA is coordinate during amino acid starvation. Therefore, we conclude that, in S. typhimurium, the synthesis of all species of stable RNA is coordinately controlled by the RC gene.
Maturation o/ 5-S RNA Amino acid starvation of an RC rel strain of E. coli lesults in the accumulation of an abnormal form of 5-S RNA e, which is a precursor of the RNA found in exponentially growing cells14. Figs. 2 and 3 show that in S. typhimurium such an abnormal 5-S RNA cannot be detected upon amino acid starvation. However, addition of chloramphenicol, either in the presence or absence of 5-methyltryptophan, did result in the accumulation of an abnormal 5-S RNA, which exhibited properties very similar to those of the E. coli 5-S RNA precursor e,14, viz. a higher affinity for a methylated albumin kieselguhr column and a lower electrophoretic mobility than mature 5-S RNA (Fig. 4). The R C rel and RC str strains gave identical results. Because of the close relationship between E. coli and S. typhimurium and the great similari*y shown by the abnormal 5-S RNA's from both organisms, the conclusion, that, in S. typhimurium also a precursor 5-S RNA exists, seems warranted. The experiments shown in Figs. 3 and 4 indicate that maturation of 5-S RNA is dependent on protein synthesis. To test this hypothesis, we studied the influence of different concentrations of chloramphenicol on maturation. Fig. 5 shows the effect /c\
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Log ~hlor0rnphenicol 3 (pg/rnl) Fig. 5. Effect of different concentrations of chloramphenicol on the R N A and protein synthesis in S. typhimurium RC str and RC tel. An exponentially growing culture of each strain w i t h an A4~0 nm of o.3, s u p p l e m e n t e d with uracil and leucine, was divided into subcultures of 2. 5 ml in 25 ml flasks. To each flask chloramphenicol was added to the desired concentration, t o g e t h e r with o. I/*C/ml of either [14C]uracil or [z4C~leucine. I n c u b a t i o n was continued for 6o min and g r o w t h was s t o p p e d b y pipetting duplicate samples of i.o ml from each flask into I.O ml of ice-cold IO % trichloroacetic acid. Samples were processed as described in MATERIALS AND METHODS and counted. Values are expressed as the percentage of the incorporation b y a control culture which did not receive chloramphenicol. , RCstr; - - - , RCrel', ('D, [[], incorporation of [z4C~uracil; O, m, incorporation of [14C]leucine.
Biochim. Biophys. Acta, 232 (1971) 314-323
320
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Fig. 6. Effect of different concentrations of chloramphenicol on the m a t u r a t i o n of 5-S R N A in
S. typhimurium RC tel. Different concentrations of chloramphenicol were added to an exponentially growing culture of the RC tel strain, and [SH]uracil 14.o FC/ml) was added 3 ° min later. I n c u b a t i o n was s t o p p e d 60 min after addition of label. Cells were harvested, mixed w i t h carrier cells, and [14C]uracil-labeled control cells a n d R N A was extracted. L o w molecular weight R N A was p r e p a r e d a n d electrophoresed on IO % (w/v) polyacrylamide gels. Panel A: 0. 5 / , g / m l chloramphenicol; Panel B: I . o p g / m l chloramphenicol. Panel C: 1. 5 f,g/ml chloramphenicol. I n the t o p of each panel the 8H/14C ratio is shown.
of chloramphenicol on the incorporation of [14C]uracil and [14Clleucine in both strains. Identical results were obtained when RNA and protein synthesis were assessed by colorimetric methods. We studied maturation of 5-S RNA at four different levels of protein synthesis, using 0.5, I.O, 1. 5 and 3.0 #g/ml of chloramphenicol. In Fig. 6 the labeling patterns, obtained after electrophoresis of low molecular weight RNA extracted from RC re1 cells treated with the three lowest concentrations of chloramphenicol mentioned, are shown. Calculation of the slope of the 3H/14C ratio curve under the 5-S peak gives a value, which is a measure of the displacement of the slower (3H) peak from the peak of control 5-S RNA, since labeling and electrophoresis were carried out under identical conditions in all cases. Table I summarizes these values,
TABLE I S L O P E O F T H E SH/14C R A T I O U N D E R T H E 5 - S P E A K , A F T E R E L E C T R O P H O R E S I S WEIGHT R~A EXTRACTED FROM Re rel CELLS, TREATED WITH DIFFERENT CHLORAMPHENICOL
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63
Biochim. Biophys. Acta, 232 (i97 I) 314-323
37
25
12
RNA SYNTHESIS IN S. typhymurium
321
obtained from experiments like those shown in Fig. 6. From these figures, we conclude that maturation of 5-S RNA can indeed take place only, if the rate of protein synthesis is above a certain level.
DISCUSSION
E//ect o/the RC gene on R N A synthesis is identical in E. coli and S. typhimurium Coordinate contlol of all species of RNA is implicated by all the models proposed to explain inhibition of RNA synthesis by amino acid starvation (see ref. I). In E. coli RC tel, synthesis of tRNA, I6-S and 23-S rRNA appear indeed to be coordinately controlled by the RC gene s, whereas there is some controversion about this with respect to E. coli RC str (see refs. 3,4). There is evidence, that synthesis of at least two species ot mRNA is not coordinate with that of stable RNA, during amino acid starvation 154~. Our results show, that in S. typhirnurium synthesis of all species of stable RNA, including 5-S RNA, is co6rdinately controlled during starvation of the R C str, a s well as the RC rd strain. There is one ieport suggesting that in S. lyphimurium, as in E. coli, mRNA synthesis is not subject to the same control by the RC gene as stable-RNA synthesis TM. The effect of the RC gene on synthesis of different species of RNA, therefore, appears to be the same in both olganisms. Nature o/the precursor 5-S R N A E. coli RC re1 cells accumulate an abnormal form of 5-S RNA during amino acid starvation, which has a higher affinity for a methylated albumin kieselguhr column and a lower electrophoretic mobility on polyacrylamide gels than 5-S RNA normally found in exponentially growing cellse. The same abnormal 5-S RNA is accumulated in wild type cells, treated with high doses of chloramphenicol (see refs. 7, 8). Fingerprinting showed a difference between normal and abnormal 5-S RNA at the 5'- end only: the latter is three nucleotides longer8. Its precursor nature for 5-S RNA, inferred from these results, was further confirmed by pulse labeling experiments in exponentially growing cells 14. The abnormal 5-S RNA, accumulated in S. typhimurium under some conditions, shows properties, which are similar to those of the E. coli precursor mentioned above. (c/. Fig. 4) It is possible to calculate roughly the molecular weight of the slower 5-S peak in Fig. 4 b from the electrophoretic mobility lg. One arrives at a figure which indicates that the abnormal 5-S RNA is only a few nucleotides longer than the mature form, which agrees with the value for the E. coli plecursor. In view of the great similarity between ~he abnormal 5-S RNA's from both olganisms, we conclude that also in S. typhimuriurn a precursor of 5-S RNA, which is slightly longer than m a t m e 5-S RNA, is synthesized. The intermediate electrophoretic mobility of the slower 5-S peak in Fig. 6b, is similar to the behaviour shown by the 5-S RNA isolated from exponentially growing E. coli by GALIBERTet al. 6. These authors conclude that there exists an "intermediate" species of 5-S RNA, which has a length of 122 nucleotides. This explanation, however, has rather complex implications with respect to the mechanism of maturation of 5-S Biochim. Biophys. Acta, 232 (1971) 314-323
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H. A. RAUI~, M. GRUBER
RNA. Our results, and probably also those of GALIBERTet al. e, can as well be explained, by the assumption that the slower peak contains about equal amounts of mature and precursor 5-S RNA. Indeed, it is possible to construct an electrophoretic pattern, which has all the characteristics of the one shown in Fig. 6b, by adding Figs. 6a and 6c.
Dependence o/maturation o/5-S R N A on protein synthesis Starvation of S. typhimurium, under our conditions, does not iesult in inhibition of maturation of 5-S RNA, as does starvation of E. colt 4,6. A precursoi of 5-S RNA does exist, however, as discussed above. It is evident from Table I, that maturation of this precursor is dependent on a minimum rate of protein synthesis. This rate, as determined by the experiments shown in Figs. 5 and 6, is between 4 ° % and 60 % of that shown by exponentially growing cells. It seems highly improbable that maturation of each preculsor 5-S RNA molecule requires a permanent at*achmept of one molecule of a specific protein. It is very unlikely, indeed, that such a relationship could be conserved, when RNA synthesis is stimulated to 13o % and protein synthesis is inhibited to 60 o~/oof the control by 0.5 #g/ml of chlotamphenicol (c/. Figs. 5 and 6). It is even more unlikely, that conservation of this relationship could be extended to the I : 4 ratio (with respect to an exponentially growing culture) of protein to RNA synthesis, shown by starved RC re1 cells. As, however, protein synthesis is a necessary condition for maturation, the protein involved must be inactivated, either by breakdown, or by removal (for instance association with a cellular organelle). It is a matter of speculation, whether this protein is the nuclease, responsible for splitting off the extra nucleotides, or a factor, which makes the three terminal nucleotides accessible to nuclease action. Amino acid starvation does not inhibit maturation From the results shown in Figs. 4 and 6, one would conclude, that the absence of inhibition of maturation during amino acid starvation, is a result of the high residual protein synthesis. However, either addition of 20/zg/ml 5-methyltryptophan, or 1. 5/~g/ml chloramphenicol, causes about the same inhibition of protein synthesis, whereas starvation has no effect on maturation. We do not have an explanation for this discrepancy, except for the hypothesis, that, although overall protein synthesis is inhibited to the same extent in both cases, synthesis of the protein necessary for maturation of 5-S RNA, is not. Unbalanced synthesis of several proteins during amino acid starvation has been described 17,2°,m. ACKNOWLEDGEMENT
We thank Mr. G. Bakker for this skillful technical assistance during part of this work. REFERENCES i G. EDLIN AND P. BRODA, Bacteriol. Rev., 32 (1968) 2o6. 2 L. R. MANDEL AND E. BOREK, Biochem. Biophys. Res. Commun., 9 (1962) I I . 3 S. SARKAR AND K. 1V[OLDAVE, .[. Mol. Biol., 33 (1968) 213.
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R N A SYNTHESIS IN
S, typhymurium
323
4 F. GALIBERT, M. E. ELADARI, C. J. LARSEN AND M. BOIRON, European J. Biochem., 13 (197 o) 273. 5 R. ROSCHENTHALER, M. A. DEVYNCK, P. FROMAGEOT AND E. j . SIMON, Biochim. Biophys., ~cta, 182 (1969) 481. 6 F. C-ALIBERT, M. E. ELADARI, A. HAMPE AND M. BOIRON, European J. Biochem., 13 (197 o) 281. 7 F. CvALIBERT, J. C. LELONG AND C. J. LARSEN, Compt. Rend., 265 (1967) 279. 8 ]3. C*. FORGET AND B. JORDAN, Science, 167 (197 o) 383 . 9 M. SCHAECHTER, O. IV~AALOE AND N. O. KJELDGAARD, J. Gen. Microbiol., 19 (1958) 592. io C. G. KURLAND AND O. MAALOE, J. Mol. Biol., 4 (1962) 193. i i R. L. SOMMERVILLE AND C. YANOFSKI, J. Mol. Biol., i i (1965) 747. 12 H. A. RAU~ AND M. GRUBER, F E B S Letters, 8 (197 o) 45. 13 R. A. WEINBERG, U. LOENING, M. WILLEMS AND S. PENMAN, Proc. Na~l. Acad. Sci. U.S., 58 (1967) lO88. 14 B. R. JORDAN, J. FEUNTEUN AND R. MONIER, J. Mol. Biol., 5 ° (197 o) 605. 15 D. W. MORRIS AND N. O. KJELDGAARD, J. Mol. Biol., 31 (1968) 145. 16 G. EDLIN, G. S. STENT, R. F. BAKER AND C. YANOFSKI, J. Mol. Biol., 37 (1968) 257. 17 R. LAVALL~ AND G. DE HAUWER, J. Mol. Biol., 37 (1968) 269. 18 P. VENETIANER, Biochem. Biophys. Res. Commun., 33 (1968) 959. 19 A. C. PEACOCK AND C. W'. DINGMAN, Biochemistry, 7 (1968) 668. 20 D. GOODMAN, H. MANOR AND W. ROMBAUTS, J. Mol. Biol., 4 ° (1969) 247. 21 D. GOODMAN, J. Mol. Biol., 51 (197 o) 491.
Biochim. Biophys. Acta, 232 (1971) 314-323
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96762
CONTROL OF STABLE-RNA S Y N T H E S I S AND M A T U R A T I O N OF 5-S RNA IN S A L M O N E L L A T Y P H I M U R I U M RC str AND RC tel
H. A. RAUI~ AND M. G R U B E R
Biochemisch Laboralorium, The University, Groningen (The Netherlands) (Received S e p t e m b e r I Ith, 197 o)
SUMMARY
I. Synthesis of stable RNA in Salmonella typhimurium RC str and RC re1 during amino acid starvation was studied with the aid of methylated albumin kieselguhr chromatography and polyacrylamide gel electrophoresis. 2. Synthesis of all classes of stable RNA was coordinately controlled by the RC gene under these conditions. The effect of the RC gene therefore appears to be the same in Escherichia coli and S. typhimurium. 3. Amino acid starvation did not result in the accumulation of an abnormal form of 5-S RNA, as in E. coli. However, addition of chloramphenicol, to either starved or exponentially growing cells, did result in the accumulation of such an abnormal form, with properties very similar to those of the E. coli 5-S RNA precursor. 4. Experiments with various low doses of chloramphenicol, indicate that maturation of 5-S RNA is dependent on a minimum rate of protein synthesis, which, under our conditions, lies between 4 ° °/o and 60 ~o of the rate shown by exponentially growing cells.
INTRODUCTION
Regulation of synthesis of stable RNA in bacteria has been widely studied since the discovery of the RC tel mutants of Escherichia coli. In contrast to the wild type (RCstr), the rate of RNA synthesis in these mutants is hardly affected b y amino acid starvation. (For a review see ref. I.) From the work of different groups 2,3, it appears that, in this organism, synthesis of t R N A and high moleculal weight RNA is coordinately controlled b y amino acid starvation (see, however, ref. 4). With respect to the control of 5-S RNA synthesis there is less information. Levallorphan inhibits synthesis of 5-S RNA and high molecular weight rRNA to the same extent, while t R N A synthesis is inhibited to a lesser extent 5. Starved E. coli RC tel cells seem to synthesize a normal amount of a precursor e as do wild type cells, treated with high doses of chloramphenicol (see refs. 7, 8). The same precursor has been found in starved RC str cells4. The effect of amino acid starvation on RNA synthesis in organisms other than E. coli has hardly been studied, probably because the only other RC re1 mutants known are those of Salmonella typhimurium. Biochim. Biophys. Acta, 232 (1971) 314-323
RNA SYNTHESIS IN S. typhimurium
315
We studied the synthesis of stable RNA, including 5-S RNA, during amino acid starvation, in two S. typhimurium strains, which, as far as is known, are isogenic except for the RC gene. In the course of these studies, we encountered an abnormal form of 5-S RNA, which showed properties similar to those of the above-mentioned E. coli 5-S RNA precursor.
MATERIALS AND METHODS
Bacterial strains Two Salmonella typhimurium strains with different control of RNA synthesis (i.e. RC str and RC re1) were obtained from Dr. A. R6rsch (Medisch Biologisch Laboratorture R.V.O.-T.N.O., Rijswijk, Nethellands). These strains were originally isolated b y Dr. A. Eisenstark and aie, as far as is known, isogenic except for the RC gene, since the relaxed strain was obtained b y bringing the RC tel gene from E. colt into wild type (RC str) S. typhimurium.
Growth conditions Cells were grown at 37 ° in flasks on a gyrotory shaker in the minimal medium described b y SCHAECHTER et al. 9, supplemented with o.2 % glycerol; 20 ffg/ml cytosine was added to reduce incorporation of uracil into DNA 1°. In measurement of RNA and protein synthesis, b y incorporation of labeled uracil or leucine, 20 ffg/ml unlabeled uracil and L-leucine were also included in the medium. Growth was followed spectropho tometrically. An overnight culture of the desired strain in minimal medium plus 0.2 % glycerol was diluted to an A450 nm of 0.03 ( I - c m path length) into fresh prewarmed medium, containing the necessary supplements. When an A45onm of 0. 3 was reached, the culture was divided into subcultures for the desiled treatment. Carrier cells were obtained b y harvesting an exponentially growing culture in the late log phase. The cells were washed once with cold Tris-MgCl 2 buffer (o.oi M Tris (pH 7.5)-O.Ol M MgCI~) and stored in aliquots of 0.6 g at --20 °.
Amino acid starvation and chloramphenicol treatment The subcultures (usually 50 ml) were subjected to amino acid starvation b y adding 20 ffg/ml 5-DL-methyltryptophan (Sigma Chem. Co., St. Louis, Mo., U.S.A.) which results in a lack of t r y p t o p h a n in the cells n. RNA was labeled b y addition of [5-3Hluracil 30 rain after addition of 5-methyltryptophan. Chloramphenicol treatment in the presence of 5-methyltryptophan, was carried out b y adding 2.5 ffg/ml chloramphenicol to a culture 30 min after addition of 5-methyltryptophan. [5-~H]Uracil was added al the same time. Chloramphenicol treatment in the absence of 5-methyltryptophan was carried out b y adding chloramphenicol to a subculture with an A45o nm of 0. 3. Incubation was continued for 30 min and [5-3Hluracil was added. In all cases incubation was stopped 60 rain after addition of the label and cells were stored at --20 ° after washing with cold Tris-MgCl 2 buffer. The amount of [3Hluracil and the concentrations of chloramphenicol used are mentioned in the legends. Biochim. Biophys. Acta, 232 (1971) 314-323
316
H . A . RAU]~, M. GRUBER
Control RNA was obtained b y labeling an exponentially growing culture of the desired strain with o.4/~C/ml [2-14C]uracil for 9 ° min from the time an A45onm of 0.3 was reached. In some cases, the label of the treated and control cells was reversed.
Measurement o] RNA and protein synthesis o.I #C/ml of either ~2-14C~uracil or L-[14Clleucine was added at time o together with 20/zg/ml 5-methyltryptophan to a subculture with an A45o nm ot 0.3. Duplicate samples of I.O ml were taken at times indicated. Samples were mixed immediately with I.O ml of ice-cold IO % trichloroacetic acid and left overnight at 4 °. Samples from a control culture were obtained at the same time. The samples were processed for counting in the usual way by filtering and washing on membrane filters (Sartorius Membranfiltergesellschaft G6ttingen, W. Germany; MF5o). Filters were counted in a Nuclear Chicago Mark I liquid scintillation counter. All values were corrected for blanks taken at time o.
RNA extraction Labeled cells from 50 ml of treated culture were mixed with labeled cells flora 50 ml of control culture and 0.6 g of carrier cells in IO ml of cold Tris-MgC12 buffer containing I.O mg/ml egg-white lysozyme (Armour Pharmaceutical Co., Eastbourne, England) and o.oi mg/ml deoxyribonuclease (Nutritional Biochem. Co. Cleveland, Ohio, U.S.A.). The suspension was frozen in liquid nitrogen and thawed at room temperature. Ariel thawing, th~ suspension was incubated for io rain in an ice bath and then sonicated in an MSE ultrasonic disintegrator for I min at 18 ooo20 ooo cycles/sec while cooled in ice. After a further incubation for 5 rain in an ice bath, RNA was extracted with 80 % (v/v) of redistilled phenol in Tris-MgC12 buffer in the presence of 0.5 % sodium dodecyl sulfate, as described ~2. The RNA was dissolved in 5.0 ml of the starting buffer for methylated albumin kieselguhr chromatography and the concentration of the solution was determined by measuring the A260 nm. The Eo~.~O~oat 260 nm was taken as 20. The recovery of RNA, as determined by measurement of radioactivity, was between 85 and 95 %.
Methylated albumin kieselguhr chromatography and preparation of low molecular weight RNA Methylated albumin kieselguhr chromatography was carried out at 35 ° as described earlier lz. Radioactivity was measured b y mixing 0.5 ml of each fraction with IO ml of scintillation fluid (300 ml dioxan, 300 ml methylcellosolve, 33 ° ml toluene, 80 g naphtalene, 4 g PPO) and counting in a liquid scintillation counter. Low moleculor weight RNA for gel electrophoresis was prepared b y adsorbing 5-6 mg of total RNA on a methylated albumin kieselguhr column in the usual way. The column was eluted with 0.6 M NaC1 in phosphate buffer. The whole procedure was carried out at room temperature to prevent denaturation of 5-S RN & which takes place only at elevated temperatures. The fluid containing A 2~0nm absorbing material was collected in a test tube cooled in ice. This solution (20--25 ml) was dialysed against 50 vol. of distilled water at 4 ° for 4 h with one change after 2 h. The residue, obtained upon lyophilization, was dissolved in the buffer used for electrophoresis, without sodium dodecyl sulfate but containing io % sucrose. The concentration of this solution, determined a s A260 nm, was adjusted to 2 mg/ml. Biochim. Biophys. Acta, 232 (1971) 314-323
RNA SYNTHESIS IN S. typhimurium
317
Polyacrylamide gel electrophoresis Electrophoresis was carried out on gels containing IO % (w/v) acrylamide (British Drug Houses, Poole, England; recrystallized from chloroform) according to the method of WEINBERG et al. z3. However, pH was kept at 7.8 and crosslinker concentration was doubled, which resulted in a slightly better resolution. Electrophoresis time was 4 h at 60 V and 32 mA using 8 gels of 9 cm length. On each gel 20/zl of a solution of low molecular weight RNA was layered. After electrophoresis, the gels were cut into I-mm slices which were hydrolysed overnight in 0.5 ml of concentrated ammonia. Ammonia was removed and radioactivity was determined by adding IO ml of scintillation fluid and counting in a liquid scintillation counter. Radiochemicals [2J4C]Uracil (specific activity 52 mC/mmole), [5-3Hluracil (specific activity 23.3 mC/mmole) and L-[14C]leucine (specific activity 311 mC/mmole, uniformly labeled), were obtained from The Radiochemical Centre, Amersham, England. RESULTS
Control o/ stable-RNA synthesis As shown in Fig. I the two strains of S. typhimurium show a behaviour characteristic of an RC s~ and RCre] genotype respectively, under conditions of amino acid ~,r M
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Fig. i. Effect of amino acid s t a r v a t i o n on R N A and protein synthesis in S. typhimurium I~Catr a n d RC tel. To an exponentially growing culture of each strain, s u p p l e m e n t e d with uracil a n d leucine, o. I / z C / m l of either [z4C]uracil or [z4C]leucine was added, t o g e t h e r w i t h 2 o / , g / m l 5-methylt r y p t o p h a n . Samples were t a k e n at t h e times indicated, filtered and w a s h e d on m e m b r a n e filters, and counted in a liquid scintillation counter. I n c o r p o r a t i o n b y a control culture was determined in the same way. Panels A and ]3: R C str, Panels C and D: RC re]. O , 5 - m e t h y l t r y p t o p h a n treated; 0 , control. Fig. 2. F r a c t i o n a t i o n of double-labeled total R N A from a m i x t u r e of an a m i n o acid s t a r v e d a n d a control culture of S. typhimurium RC str, b y m e t h y l a t e d a l b u m i n kieselguhr c h r o m a t o g r a p h y . [3H]Uracil (8.0 #C/ml) was added to a culture of the RC str strain 3 ° min after t h e addition of 5 - m e t h y l t r y p t o p h a n . Cells were h a r v e s t e d 60 m i n later, mixed w i t h carrier cells, and [14CJuracillabeled control cells, a n d extracted w i t h phenol. I~NA was c h r o m a t o g r a p h e d on a m e t h y l at a l b u m i n kieselguhr c o l u m n (2.1 cm × 12 cm) a t 35 °.
Biochim. Biophys. Act(*, 232 (197I) 314-323
318
H . A . RAUE, M. GRUBER
starvation. Protein synthesis is inhibited to about the same extent in both strains. RNA synthesis in the RC str cells is depressed to about 20 %; in the RC rd cells no reduction in RNA synthesis can be detected up to 9 ° min after addition of 5-methyltryptophan. The residual protein synthesis is rather high in comparison to t h a t observed in auxotrophic strains during amino acid starvation. Further reduction, however, by increasing the concentration of 5-methyltryptophan, was impossible. Therefore, a concentration of 20#g/ml was routinely used. To study the synthesis of different species of RNA during amino acid starvation, chromatography on methylated albumin kieselguhr columns was used for a rough separation. Fig. 2 shows a typical chromatogram of double-labeled total RNA from amino acid starved and control RC str cells. A portion of the 5-S RNA is denatured on the column under the conditions used, hence this RNA is eluted in two peaks ~2. The labeling pattern of RNA isolated from a mixture of starved and control RC r~l cells was essentially the same, except for differences in the 23-S peak which are now under investigation. Since the recovery of RNA during isolation and chlomatography in most cases was better than 9 ° °/o, the labeling pattern allows us to conclude, that synthesis of t R N A and high molecular weight rRNA in S. typhimurium is coordinately controlled b y amino acid starvation. The same conclusion probably holds true for synthesis of 5-S RNA, but it is difficult to infer this unambiguously from the methylated albumin kieselguhr chromatogram, because of the incomplete resolution of the 5-S peaks. Therefore, we used polyacrylamide gel electrophoresis, which allows an almost complete separation of 5-S RNA and tRNA.
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Fig. 3. Synthesis of 5-S R N A b y S. typhimurium :RCstr and RC re1 during amino acid starvation. Low molecular weight R N A was p r e p a r e d from a m i x t u r e of [DH]uracil-labeled starved, and [14C]uracil-labeled control cells of either strain. I n the case of the RC str strain 8.0/~C/ml ~eH]uracil was used, in t h a t of the RC tel strain 4.o #C/ml. The R N A was electrophoresed on IO % (w/v) polya6rylamide gels. Gels were sliced, hydrolysed, and counted in a liquid scintillation counter. Panel A: RC str. Panel B: RC tel. I n the top of each panel the SH/x4c ratio is shown. Fig. 4- Synthesis of 5-S R N A in S. typhimurium RC tel, t r e a t e d with chloramphenicol in t h e presence of 5 - m e t h y l t r y p t o p h a n . Chloramphenicol (2. 5/~g/ml) was added to a culture of the RC rel strain, together with 0. 4 #C/ml [14C]uracil, 3 ° min after the addition of 5 - m e t h y l t r y t o p h a n . Cells were harvested 60 rain later, mixed with carrier cells, and control cells labeled with 4.0 I,C/ ml ESH]uracil and extracted with phenol. A p o r t i o n of the R N A obtained, was used for methylated a l b u m i n kieselguhr c h r o m a t o g r a p h y . L o w molecular weight RlqA was p r e p a r e d from the other portion, and electrophoresed on io % (w]v) polyacrylamide gels. Panel A: m e t h y l a t e d a l b u m i n kieselguhr c h r o m a t o g r a m ; only t h e first p a r t of the labeling p a t t e r n is shown. Panel B: electrophoretic p a t t e r n .
Biochim. Biophys. Acta, 232 (1971) 314-323
RNA SYNTHESIS IN S. typhimurium
319
Fig. 3 shows the electrophoretic pattern of low-molecular weight RNA: isolated from a mixture of starved and control cells of either strain. It is clear, that in either case, the ratio of 5-S RNA to tRNA is the same in starved and in exponentially growing cells, which indicates that synthesis of both species of RNA is coordinate during amino acid starvation. Therefore, we conclude that, in S. typhimurium, the synthesis of all species of stable RNA is coordinately controlled by the RC gene.
Maturation o/ 5-S RNA Amino acid starvation of an RC rel strain of E. coli lesults in the accumulation of an abnormal form of 5-S RNA e, which is a precursor of the RNA found in exponentially growing cells14. Figs. 2 and 3 show that in S. typhimurium such an abnormal 5-S RNA cannot be detected upon amino acid starvation. However, addition of chloramphenicol, either in the presence or absence of 5-methyltryptophan, did result in the accumulation of an abnormal 5-S RNA, which exhibited properties very similar to those of the E. coli 5-S RNA precursor e,14, viz. a higher affinity for a methylated albumin kieselguhr column and a lower electrophoretic mobility than mature 5-S RNA (Fig. 4). The R C rel and RC str strains gave identical results. Because of the close relationship between E. coli and S. typhimurium and the great similari*y shown by the abnormal 5-S RNA's from both organisms, the conclusion, that, in S. typhimurium also a precursor 5-S RNA exists, seems warranted. The experiments shown in Figs. 3 and 4 indicate that maturation of 5-S RNA is dependent on protein synthesis. To test this hypothesis, we studied the influence of different concentrations of chloramphenicol on maturation. Fig. 5 shows the effect /c\
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Log ~hlor0rnphenicol 3 (pg/rnl) Fig. 5. Effect of different concentrations of chloramphenicol on the R N A and protein synthesis in S. typhimurium RC str and RC tel. An exponentially growing culture of each strain w i t h an A4~0 nm of o.3, s u p p l e m e n t e d with uracil and leucine, was divided into subcultures of 2. 5 ml in 25 ml flasks. To each flask chloramphenicol was added to the desired concentration, t o g e t h e r with o. I/*C/ml of either [14C]uracil or [z4C~leucine. I n c u b a t i o n was continued for 6o min and g r o w t h was s t o p p e d b y pipetting duplicate samples of i.o ml from each flask into I.O ml of ice-cold IO % trichloroacetic acid. Samples were processed as described in MATERIALS AND METHODS and counted. Values are expressed as the percentage of the incorporation b y a control culture which did not receive chloramphenicol. , RCstr; - - - , RCrel', ('D, [[], incorporation of [z4C~uracil; O, m, incorporation of [14C]leucine.
Biochim. Biophys. Acta, 232 (1971) 314-323
320
H . A . RAU~, M. GRUBER
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Fig. 6. Effect of different concentrations of chloramphenicol on the m a t u r a t i o n of 5-S R N A in
S. typhimurium RC tel. Different concentrations of chloramphenicol were added to an exponentially growing culture of the RC tel strain, and [SH]uracil 14.o FC/ml) was added 3 ° min later. I n c u b a t i o n was s t o p p e d 60 min after addition of label. Cells were harvested, mixed w i t h carrier cells, and [14C]uracil-labeled control cells a n d R N A was extracted. L o w molecular weight R N A was p r e p a r e d a n d electrophoresed on IO % (w/v) polyacrylamide gels. Panel A: 0. 5 / , g / m l chloramphenicol; Panel B: I . o p g / m l chloramphenicol. Panel C: 1. 5 f,g/ml chloramphenicol. I n the t o p of each panel the 8H/14C ratio is shown.
of chloramphenicol on the incorporation of [14C]uracil and [14Clleucine in both strains. Identical results were obtained when RNA and protein synthesis were assessed by colorimetric methods. We studied maturation of 5-S RNA at four different levels of protein synthesis, using 0.5, I.O, 1. 5 and 3.0 #g/ml of chloramphenicol. In Fig. 6 the labeling patterns, obtained after electrophoresis of low molecular weight RNA extracted from RC re1 cells treated with the three lowest concentrations of chloramphenicol mentioned, are shown. Calculation of the slope of the 3H/14C ratio curve under the 5-S peak gives a value, which is a measure of the displacement of the slower (3H) peak from the peak of control 5-S RNA, since labeling and electrophoresis were carried out under identical conditions in all cases. Table I summarizes these values,
TABLE I S L O P E O F T H E SH/14C R A T I O U N D E R T H E 5 - S P E A K , A F T E R E L E C T R O P H O R E S I S WEIGHT R~A EXTRACTED FROM Re rel CELLS, TREATED WITH DIFFERENT CHLORAMPHENICOL
Expt.
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~H/z4C ratio Chloramphenicol concn. ~ug/ml) : 0.5
z.o
L5
3.o
I II III IV
0.9 0.4 0.8 --
2.9 3 .2 3.0 3.3
6.0 5.5 5.6 5 .1
7.4 5-3 5.3 4 .8
Mean value
0.7
3.1
5.6
5.7
Protein synthesis (% of exptl, growing control)
63
Biochim. Biophys. Acta, 232 (i97 I) 314-323
37
25
12
RNA SYNTHESIS IN S. typhymurium
321
obtained from experiments like those shown in Fig. 6. From these figures, we conclude that maturation of 5-S RNA can indeed take place only, if the rate of protein synthesis is above a certain level.
DISCUSSION
E//ect o/the RC gene on R N A synthesis is identical in E. coli and S. typhimurium Coordinate contlol of all species of RNA is implicated by all the models proposed to explain inhibition of RNA synthesis by amino acid starvation (see ref. I). In E. coli RC tel, synthesis of tRNA, I6-S and 23-S rRNA appear indeed to be coordinately controlled by the RC gene s, whereas there is some controversion about this with respect to E. coli RC str (see refs. 3,4). There is evidence, that synthesis of at least two species ot mRNA is not coordinate with that of stable RNA, during amino acid starvation 154~. Our results show, that in S. typhirnurium synthesis of all species of stable RNA, including 5-S RNA, is co6rdinately controlled during starvation of the R C str, a s well as the RC rd strain. There is one ieport suggesting that in S. lyphimurium, as in E. coli, mRNA synthesis is not subject to the same control by the RC gene as stable-RNA synthesis TM. The effect of the RC gene on synthesis of different species of RNA, therefore, appears to be the same in both olganisms. Nature o/the precursor 5-S R N A E. coli RC re1 cells accumulate an abnormal form of 5-S RNA during amino acid starvation, which has a higher affinity for a methylated albumin kieselguhr column and a lower electrophoretic mobility on polyacrylamide gels than 5-S RNA normally found in exponentially growing cellse. The same abnormal 5-S RNA is accumulated in wild type cells, treated with high doses of chloramphenicol (see refs. 7, 8). Fingerprinting showed a difference between normal and abnormal 5-S RNA at the 5'- end only: the latter is three nucleotides longer8. Its precursor nature for 5-S RNA, inferred from these results, was further confirmed by pulse labeling experiments in exponentially growing cells 14. The abnormal 5-S RNA, accumulated in S. typhimurium under some conditions, shows properties, which are similar to those of the E. coli precursor mentioned above. (c/. Fig. 4) It is possible to calculate roughly the molecular weight of the slower 5-S peak in Fig. 4 b from the electrophoretic mobility lg. One arrives at a figure which indicates that the abnormal 5-S RNA is only a few nucleotides longer than the mature form, which agrees with the value for the E. coli plecursor. In view of the great similarity between ~he abnormal 5-S RNA's from both olganisms, we conclude that also in S. typhimuriurn a precursor of 5-S RNA, which is slightly longer than m a t m e 5-S RNA, is synthesized. The intermediate electrophoretic mobility of the slower 5-S peak in Fig. 6b, is similar to the behaviour shown by the 5-S RNA isolated from exponentially growing E. coli by GALIBERTet al. 6. These authors conclude that there exists an "intermediate" species of 5-S RNA, which has a length of 122 nucleotides. This explanation, however, has rather complex implications with respect to the mechanism of maturation of 5-S Biochim. Biophys. Acta, 232 (1971) 314-323
322
H. A. RAUI~, M. GRUBER
RNA. Our results, and probably also those of GALIBERTet al. e, can as well be explained, by the assumption that the slower peak contains about equal amounts of mature and precursor 5-S RNA. Indeed, it is possible to construct an electrophoretic pattern, which has all the characteristics of the one shown in Fig. 6b, by adding Figs. 6a and 6c.
Dependence o/maturation o/5-S R N A on protein synthesis Starvation of S. typhimurium, under our conditions, does not iesult in inhibition of maturation of 5-S RNA, as does starvation of E. colt 4,6. A precursoi of 5-S RNA does exist, however, as discussed above. It is evident from Table I, that maturation of this precursor is dependent on a minimum rate of protein synthesis. This rate, as determined by the experiments shown in Figs. 5 and 6, is between 4 ° % and 60 % of that shown by exponentially growing cells. It seems highly improbable that maturation of each preculsor 5-S RNA molecule requires a permanent at*achmept of one molecule of a specific protein. It is very unlikely, indeed, that such a relationship could be conserved, when RNA synthesis is stimulated to 13o % and protein synthesis is inhibited to 60 o~/oof the control by 0.5 #g/ml of chlotamphenicol (c/. Figs. 5 and 6). It is even more unlikely, that conservation of this relationship could be extended to the I : 4 ratio (with respect to an exponentially growing culture) of protein to RNA synthesis, shown by starved RC re1 cells. As, however, protein synthesis is a necessary condition for maturation, the protein involved must be inactivated, either by breakdown, or by removal (for instance association with a cellular organelle). It is a matter of speculation, whether this protein is the nuclease, responsible for splitting off the extra nucleotides, or a factor, which makes the three terminal nucleotides accessible to nuclease action. Amino acid starvation does not inhibit maturation From the results shown in Figs. 4 and 6, one would conclude, that the absence of inhibition of maturation during amino acid starvation, is a result of the high residual protein synthesis. However, either addition of 20/zg/ml 5-methyltryptophan, or 1. 5/~g/ml chloramphenicol, causes about the same inhibition of protein synthesis, whereas starvation has no effect on maturation. We do not have an explanation for this discrepancy, except for the hypothesis, that, although overall protein synthesis is inhibited to the same extent in both cases, synthesis of the protein necessary for maturation of 5-S RNA, is not. Unbalanced synthesis of several proteins during amino acid starvation has been described 17,2°,m. ACKNOWLEDGEMENT
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