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The formation of “ribosomal RNA” in escherichia coli during recovery from magnesium starvation
610
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95054
T H E F O R M A T I O N OF " R I B O S O M A L R N A " IN E S C H E R I C H I A
COLI
DURING RECOVERY FROM MAGNESIUM STARVATION HIDEHO
S U Z U K I ~ AWl) Y U K I M A S A H A Y A S H I
Institute o[ Molecular Biology, Faculty o[ Science, Nagoya University, Chikusa-Ku, Nagoya (Japan) (Received F e b r u a r y 12th, I964)
SUMMARY
I. When Escherichia coli cells are incubated in a medium lacking magnesium, they lose most of their ribosomes without loss of viability, as reported by McCARTHY1. 2. Upon the addition of magnesium, the cells accumulate "nascent ribosomal RNA" in the particles which appear to correspond to those synthesized in the presence of chloramphenicol. 3. The nascent ribosomal RNA has a slightly different sedimentation coefficient from that of the mature ribosomal RNA. This is probably due to the difference in secondary structure between these two types of RNA, a difference which can be shown by the hyperchromicity of the nascent form upon heating.
INTRODUCTION
It is known that, when cells of Escherichia coli are incubated in a growth medium lacking magnesium, they lose most of their original ribosomes without loss of viability 1. Upon the addition of magnesium such "ribosome-free" E. coli gradually resume the synthesis of ribosomal particles before the initiation of normal growth. The synthesis of ribosomal particles and r-RNA, in the early phase of recovery from magnesium-starvation, has been investigated in the present study. It has been found that, immediately after magnesium replenishment, the "ribosome-free" cells accumulate nascent r-RNA in unusual particles without the synthesis of typical ribosomes for a long period. These particles appear to correspond to those synthesized in the presence of chloramphenicol. Since such cells have neither pre-formed nor newly synthesized, typical ribosomes, a tangible quantity of the nascent r-RNA can be isolated. Direct chemical analysis shows that this RNA has a ribosomal type of ratio for its component bases. However, it possesses a sedimentation coefficient which is slightly different from typical r-RNA and also it has less hyperchromicity upon heating. Abbreviation: r-RNA, ribosomal RNA. " P r e s e n t address: N a t i o n a l I n s t i t u t e of Genetics, M i s h i m a ( J a p a n ) .
Biochim. Biophys. Acta, 87 (1964) 6 1 o - 6 2 o
FORMATION OF RIBOSOMAL RNA
611
MATERIALS AND METHODS
Bacterial culture Usually one loopful of E. coli B(I-I) cells were inoculated into 50 ml of Trisglucose medium 2. After overnight incubation the cells were transferred to fresh medium of the same composition to give a bacterial concentration of 1. 5. lO s cells/ml. They were then reincubated over 3 generations. The cells were collected and washed twice with magnesium-free medium (or saline) and then suspended in Tris-glucose medium lacking magnesium. The magnesium starvation was performed by culturing the bacteria under powerful aeration for 20 h. At the end of this period, viable count did not change, and the ribosomal content of the cell was decreased to a minimum, as described by ]V[CCARTHY1. The medium for the restoration from magnesiumstarvation contained o.I % casamino acids in addition to the usual Tris-glucose medium. This medium will be designated as the "restoration medium" throughout this paper.
Incorporation o~ isotopes For the measurement of isotope-incorporation into the RNA and the protein of whole cells, the following procedure was employed. Of two flasks (each containing 20 ml of the restoration medium), one (A) received 5#C of ~1~C]uracil (6.5/~C/ mmole), the other (B) 3.7 #C of [14C]leucine (7.41/*C/mg), and magnesium-starved cells to give 4" los cells per millilitre. Samples of 2 ml each were taken out after an appropriate interval of time and added (A) to 2 ml of cold IO % trichloroacetic acid in an ice bath, or (B) to 2 ml of hot trichloroacetic acid at 90° in a water bath in which case they were cooled after 15 rain. The precipitate was collected on a ultrafilter 984H (Hurlbut Paper Co. (U.S.A.)), washed with 5 % trichloroacetic acid, and then dried. The radioactivity was measured in a Geiger counter. For the preparation of the sample for sedimentation-analysis, El~C]uracil (o.I ~C) was added to IOO ml of medium at the outset of incubation.
Preparation o~ crude cell-extract Harvested cells were washed with o.oi M Tris buffer (pH 7.8) containing o.oooi M magnesium acetate, centrifuged and the pellet was then frozen immediately at --20 °. The frozen cells were ground with fine quartz sand for 2-3 rain and extracted with about 3 volumes of the above buffer. Sand and the cell debris were removed b y centrifugation at 7500 rev./min for IO rain. Deoxyribonuclease (EC 3.1.4.5) (DNAase, twice crystallized and obtained from Worthington Biochemical Corp., N.J. (U.S.A.)) was added, at a concentration of 5/~g/ml, at this stage.
Preparation o~ RNA The procedure employed here was essentially the same as that described previously 3. Bacterial cells were ground with quartz sand in the presence of sodium lauryl sulphate and extracted with 0.005 M Tris buffer (plK 7.8) containing 0.2 % sodium lauryl sulphate. The extract was shaken for 3 rain with an equal volume of 9 ° % (w/w) phenol in the cold. The aqueous layer was taken out, and another volume of Tris-sodium lauryl sulphate was added to the phenolic phase. The extraction was Biochim. Biophys. Acta, 87 (1964) 61o-62o
012
H. SUZUKI AND Y. HAYASHI
repeated. From the combined aqueous layer, the nucleic acids were precipitated with 2 volumes of cold ethanol, redissolved in o.o2 M acetate buffer - o . I M NaC1 (pH 5.o), and dialyzed against the same buffer for about 2 h. They were finally treated with DNAase (4 #g/ml).
Sucrose density-gradient centrifugation The crude cell extract was layered on to 27 ml of sucrose (5 % to 20 O//ogradient) containing the same Tris buffer as for the preparation of the cell extract, and was centrifuged using a Spinco L SW 25.1 rotor at 23 ooo or 25 ooo rev./min. For the fractionation of RNA, a gradient of 2.5 % to I5 % of sucrose 4 in 0,05 M acetateo.I M NaC1 (pH 5.0) was used. This centrifugation was performed using the same rotor at 25 ooo rev./min for 13 h. Io-15 drops were collected by dripping from the bottom of the tube. After the measurement of 1260rn/*, RNA was precipitated with cold 8 % trichloroacetic acid together with carrier protein. The precipitate was centrifuged, washed twice with 5 o//otrichloroacetic acid, dissolved in dilute ammonia solution, and then dried on a steel planehet. The radioactivity was measured either in a Geiger counter, or a gas-flow counter. In order to isolate separately either the I6-(I7) or the 23-(22.5) -S RNA component, the RNA preparation was subjected to the sucrose density-gradient centrifugation as described above. Two volumes of ethanol were added to precipitate the R N A and the precipitate was stored at --20 ° until use.
Nucleotide composition and chemical analysis Nucleotide composition was analyzed by the Dowex-i column chromatographic method as described b y OSAWA et al. ~. RNA was assayed by the orcinol reaction and protein by LOWRY'S method.
A bsorbancy-temperature profile The hyperchromicity of RNA at 260 m# was determined at different temperatures in 0.o05 M phosphate buffer (pit 6.3) containing I M NaC1 and using a thermostatically controlled spectrophotometer (Hitachi EPU). RESULTS
Disappearance of ribosomes in magnesium-starved cells The magnesium-starved cells lost practically all of the ribosomes of the types 30 S and 50 S (Fig. I), and contained a component with sedimentation coefficient of about 25 S. In order to estimate the amount of typical ribosomal particles which might still remain, the exponentially growing cells were labelled with 32P1 (o.oi mC/ml of culture medium). The cells were then washed and incubated in the absence of magnesium. After incubation, an extract was made from the cells in o.oooi M magnesium acetate and it was analyzed b y sucrose density-gradient centrifugation. From this experiment, the ribosomal content was estimated to be about 1. 3 % of the normally grown cells as judged b y the ~*P content in the 5o-S ribosome region. Larger particles (7° S and IOO S) were not observed in a sample derived from the medium containing a higher concentration (o.oi M ) o f magnesium.
Biochim. Biophys. Acta, 87 (1964) 61o-62o
FORMATION OF RIBOSOMAL R N A 50S
1.5
613
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Fig. i. Absence of ribosomes in crude extract from magnesium-starved cells, a, exponentially growing cells; b, magnesium-starved cells. Sucrose density-gradient, 5 % to 20 % in o.oooi M magnesium acetate-o.oi M Tris buffer (pH 7.8). Centrifugation for IO h at 23 ooo rev./min. Total amount of extract from approx. 3" 1°1° cells was applied.
Nucleic acids were prepared by the "phenol" method from magnesium-starved cells and these were subjected to sucrose density-gradient centrifugation. The bulk of A~60m/z absorbing material was detected in 4-1o S with only a trace of it in the 23-S region (Fig. 2b), where the typical r-RNA should reside (Fig. 2a). The results described in this section are in essential agreement with those reported by ]V[CCARTHY1.
Growth, and synthesis o/ RNA and o/ protein during the recovery /rom magnesiumstarvation A~50 m~ of t h e c u l t u r e in t h e r e s to r a t io n m e d i u m r e m a i n e d c o n s t a n t for m o r e t h a n 2 h. A t least 3 h were r e q u i r e d u n ti l the cells r e s u m e d a l o g a r i t h m i c growth. T h e first t race of ty p i c a l 3o-S a n d 5o-S ribosomes usually a p p e a r e d 2-3 h after t h e a d d i t i o n of m a g n e s i u m .
Biochim. Biophys. Acta, 87 (1964) 6Io-62o
614
H. SUZUKI AND Y. HAYASHI 23S
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10 20 30 Tube n u m b e r
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Fig. 2. Absence of typical r-RNA in magnesium-starved cells, a, exponentially growing cells; b, magnesium-starved cells. Sucrose density-gradient, 2.5 % to 15 % in o.o5M magnesium acetate-o.I M NaCI (pH 5.0). Centrifugation at 25 ooo rev./min for 13 h.
RNA 3o0
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Fig. 3. Protein and RNA synthesis during the course of recovery from magnesium-starvation. a, chemical measurements; b, incorporation of isotopes.
Biochim. Biophys. Acla, 87 (1964) 6Io-62o
FORMATION O F RIBOSOMAL
RNA
6i 5
As shown in Figs. 3a and 3b, the synthesis of RNA as measured either by direct chemical determination or by [14C]uracilincorporation into RNA, started immediately after the addition of magnesium. On the other hand the synthesis of protein was strongly inhibited for about 3 h, as seen by the chemical analyses and by the incorporation of [~dC]leucine into protein. The synthesis then gradually recovered its normal rate.
Properties o/ the particles containing RNA synthesized in the early phase o/magnesiumreplenishment Crude extracts from the cells exposed to [14C]uracil for different periods of time were analysed by sucrose density-gradient centrifugation. With exposure for IO rain, most of the activity was detected as 24-25-S and I7-I8-S particles (Fig. 4a). The prolonged incubation (for 9 ° rain and 18o rain) in the restoration medium allowed these particles to accumulate in a considerable amount (Figs. 4b and 4c). The radioactivity peaks coincided with those manifested by A~e0 m#. These unusual particles 50S
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Fig. 4. S e d i m e n t a t i o n - a n a l y s e s of c r u d e e x t r a c t labelled w i t h [14C]uracil. E x p o s e d to [14C]uracil (o.oi #C) for: a, io m i n ; b, 90 rain; c, i 8 o m i n . Sucrose d e n s i t y - g r a d i e n t , t h e s a m e as in Fig. I. C e n t r i f u g a t i o n a t 25 ooo r e v . / m i n for: a, 9 h; b, c 7 h. A,80m v of 3o-S a n d 5o-S p e a k s are d u e to n o r m a l r i b o s o m e s a d d e d as reference.
Biochim. Biophys. Acta, 87 (1964) 6:to-62o
010
H. SUZUKI AND Y. HAYASHI
could be observed over a long period of time, and they were still present after the cells had started to grow exponentially. From the distribution of radioactivity in Figs. 4b and 4c, it is apparent that a considerable amount of 4-S RNA (probably soluble RNA) was also synthesized besides the particles mentioned above. Chloramphenicol is known to inhibit the synthesis of protein, and it accumulates "chloramphenicol-particles" (18 S and 25 S) when added to the cells which have been grown normallyd, 7. In the case of magnesium-replenishment also, the protein synthesis is strongly suppressed, together with the accumulation of the unusual particles. We therefore compared the sedimentation pattern of our unusual particles with that of chloramphenicol particles by the use of the analytical ultracentrifuge. For the preparation of chloramphenicol-inhibited cells, exponentially growing E. coli cells were incubated for 1.5 h with 200 ug of chloramphicol. The collected cells were used for the preparation of crude extract. Typical chloramphenicol-particles with sedimentation coefficients of 19 S and 15 S (uncorrected) could be seen in Fig. 5a in b
7_0
Chlor'amphenicol
tD
O~
a Mg2+ replenished
O
'
/ Fig. 5. Sedimentation-analyses of crude extracts from chloramphenicol-inhibited cells and Iron1 magnesium-replenished ceils. a, chloramphenicol-inhibited cells; b, magnesium-replenished cells. Photographs taken 16 rain after reaching 5° 74° rev./min. Solvent: o.oooi M magnesium acetate-o.I M Tris buffer (pH 7.8). the case of chloramphenicol-inhibited cells. The pattern of magnesium-replenished cell-extract is shown in Fig. 5b and is essentially the same as that of Fig. 5a, except t h a t no 50 S and 3o S peaks were seen in the latter.
Properties o/ the RNA synthesized in the early phase o/ magnesium-replenishment Magnesium-starved cells were labelled with [14Cluracil in the restoration medium for IO rain. They were mixed with an approximately equal amount of nnlabelled normal cells as the source of standard I6-S and 23-S r-RNA. RNA was then prepared by the "phenol" method and centrifuged in a sucrose density-gradient. The labelled RNA appeared in the 23-S and the I6-S regions and some in the 4-S region (Fig. 6). As deduced from the pattern of the crude extract (Fig. 4a), the radioactive RNA in the I6-S-23-S region was probably derived from the I7-S-25-S particles. The RNA labelled '16 S' showed a tendency to go ahead of the standard 16 S, and the
Biochim. Biophys. Acta, 87 (1964) 61o-6zo
FORMATION OF RIBOSOMAL
RNA
617
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/ 200
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ki 100 "o~ 0
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30
Fig. 6. S e d i m e n t a t i o n - a n a l y s e s of R N A labelled w i t h [14C]uracil. E x p o s e d to [z4C]uracil for IO m i n . Sucrose d e n s i t y - g r a d i e n t , t h e s a m e as in Fig. 2. C e n t r i f u g a t i o n at 25 ooo r e v . / m i n for 13 h.
'23 S' behind the 23 S. This pattern is similar to the case of nascent r-RNA reported by ~/IITSUI et al. ~. Since the RNA was accumulated in the I7-S-25-S particles for a long period without the formation of typical 3o-S and 5o-S particles, and since no pre-formed particles (therefore no I6-S and 23-S r-RNA) were present in the cells, the r-RNA which was newly synthesized upon magnesium-replenishment could be obtained in quantity, which is directly observable by A260m# measurement. Magnesium-starved cells were incubated in the restoration medium for 9 ° rain without isotope, and the RNA was prepared by the "phenol" method. The preparation was mixed with a trace amount of s2P-labelled authentic I6-S and 23-S r-RNA, and centrifuged in a sucrose density gradient. The obtained pattern indicated just the reverse situation between absorbancy and radioactivity as compared with the preceding 14C-experiment (Fig. 7). The Az60m/~ of the newly formed r-RNA was slightly behind that of 23S
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Fig. 7. S e d i m e n t a t i o n - a n a l y s i s of R N A prepared f r o m cells i n c u b a t e d for 90 rain after the a d d i t i o n of m a g n e s i u m . Sucrose d e n s i t y - g r a d i e n t , t h e s a m e as in Fig. 2. C e n t r i f u g a t i o n at 25 ooo r e v . / m i n . A t r a c e of s2P-labelled a u t h e n t i c r - R N A f r o m n o r m a l l y g r o w i n g cells w a s added as t h e reference.
Biochim. Biophys. Acta, 87 (1964) 6 1 o - 6 2 o
0I~
H. SUZUKI AND Y. HAYASHI
[a2P]r-RNA in the I6-S region, and slightly ahead in the 23-S region. From this experiment, we could conclude that the slight difference in sedimentation coefficients noted in the l~C-experiment between the nascent r-RNA and the authentic r-RNA was a reality; and that it was not occasioned by an aggregation of smaller components with some of the r-RNA molecules. The nascent r-RNA in question was estimated to have sedimentation coefficients of both 17 S and 22.5 S, in accordance with those of pulse-labelled nascent r-RNA under the 'shift-up' condition reported by MIa'SuI et al. a.
The results of the base-ratio analyses on tile nascent r-RNA and the authentic r-RNA are presented in Table I. From the results, it is clear that the nascent r-RNA synthesized in the early stage of recovery from magnesium-starvation bore tile ribosomal type base-ratio. TABLE I NUCLEOTIDE COMPOSITION OF 'NASCENT RIBOSOMAL RN.X_' FROM MAGNESIUM-REPLENISHED CELLS Total R N A was p r e p a r e d from cells 12o rain after the addition of magnesium. ' N a s c e n t ribosomal R N A ' was t h e n separated f r o m low molecular weight R N A b y precipitation in 2 M NaC1. Sedi m e n t a t i o n - a n a l y s i s of the R N A so p r e p a r e d indicated t h a t it is m a i n l y composed of I7-S and 22.5-S RNA.
' N a s c e n t ribosomal R N A ' Ribosomal RNA
.4 denylic acid
Guanylic acid
Cytidylic acid
Uridylic acid
24. 4 24.i
31,4 31.9
21.i 21. 5
23.o 22.8
The data of hyperchromicity upon heating of the newly formed I7-S and 22.5-S r-RNA are shown in Figs. 8a and 8b, in comparison with those of authentic I6-S and 23-S r-RNA. All the RNA components used were isolated by sucrose densitygradient centrifugation from total RNA preparation. Each sample so prepared gave a single peak on sedimentation analysis, with a slight shoulder of the other material. Both 17 S and 22.5 S, especially 22.5 S, revealedhyperchromic effect to a~considerably lower extent than authentic I6-S and 23-S r-RNA respectively. DISCUSSION
MCCARTHY1 reported that, when E. coli cells are subjected to magnesium-starvation, they are depleted of preformed ribosomes. We have now found that, upon replenishment of magnesium ions to such cells, tile synthesis of what appeared to be the "nascent" or "premature" r-RNA (17 S and 22.5 S) having a ribosomal type of base-ratio is immediately resumed, while the synthesis of protein is suppressed for a long period. The nascent r:RNA is incorporated into particles which appear to correspond to chloramphenicol-particles 6& The situation therefore parallels that prevailing when bacteria are treated with chloramphenicol. A slight difference between the sedimentation coefficients of nascent and authentic r-RNA might be caused by the difference in their chain length. It is probable that the nascent r-RNA has the size corresponding to the mature r-RNA already in their incipient synthetic stage, and the difference in sedimentation coefficients is due to the difference in secondary structure. Although the evidence is not yet conclusive Biochim.
Biophys,
Acla,
87 (1964) 61o-62o
FORMATION OF RIBOSOMAL RNA
619
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20
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30
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70
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Temperature
b 2c
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40 510 6() "ZO dO Tem perature Fig. 8. U l t r a v i o l e t absorbancy-temperature profile of mature and nascent r - R N A , a, I6-S r - R N A a n d I7-S n a s c e n t r - R N A ; b, 23-S r - R N A and 22.5-S n a s c e n t r-RNA.
on this point, both I7-S and 22.5-S r-RNA exhibited a significantly lower hyperchromicity upon heating than I6-S and 23-S r-RNA, thus suggesting that the nascent r-~RNA has less secondary structure than the I6-S and 23-S r-RNA. Nascent r-RNA detected b y pulse-labelling of the 'shift-up' cells also has sedimentation coefficients of 17 S and 22 S (see ref. 3). It forms a rapidly sedimenting aggregate much more readily than r-RNA at higher concentrations of magnesium s, which also suggests a difference in the secondary structure. NOTE ADDED IN PROOF
Experiments were performedrecently on the sedimentation behaviour of the nascent and mature r-RNA at near infinite dilution. Peaks were identified b y SH and 14C labels in a Tri-carb Liquid Scintillation Spectrometer. Tile behaviour of the minor component (16-17 S) aof 3H-labelled nascent r-RNA was, as shown in the present paper, definitely different from that of the mature one labelled with 14C. However, no difference was detected on the major component of nascent and mature r-RNA under the same conditions. Received August 7th, 1964 ACKNOWLEDGEMENTS
The authors wish to thank Dr. S. OSAWAfor much help throughout this study. Some of the experiments were performed at the Research Institute for Nuclear Medicine Biochim. Biophys. Acta, 87 (1964) 61o-62o
620
H. SUZUKI AND Y. HAYASHI
and Biology, Hiroshima University, where Professor A. SIBATANI gave us much fruitful advice. The work was supported by grants from the U.S. Public Health Service to Dr. OSAWA (GM lO466-Ol ), the Asahi Press and the Ministry of Education of Japan. REFERENCES 1 g . J. MCCARTHY, Biochim. Biophys. Acla, 55 (1962) 88o. A. ISHIHAMA, N. MIZUNO, M. TAKAI, E. OTAKA AND S. OSAWA,J. l~/Iol. BioL, 5 (1962) 251. 3 H. MITSUI, A. ISHIHAMA AND S. OSAWA, Biochim. Biophys. Acta, 76 (1963) 4Ol. * M. HAYASHI AND S. SPIEGELMAN,Proc. Natl. Acad. Sci., U.S., 47 (1961) 1564. 5 S. OSAWA, •. TAKATA AND Y. HOTTA, Biochim. Biophys. Acta, 28 (1958) 271. * M. NOMURA AND J. D. WATSON, J. Mol. Biol., i (1959) 2o4. C. G. KURLAND, M. NOMURA AND J. D. WATSON', J. Mol. Biol., 4 (1962) 388. 8 S. OSAWA, Biochim. Biophys. Acta, 42 (196o) 244.
Biochim. Biophys. Acta, 87 (1964) 61o-62o
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 95054
T H E F O R M A T I O N OF " R I B O S O M A L R N A " IN E S C H E R I C H I A
COLI
DURING RECOVERY FROM MAGNESIUM STARVATION HIDEHO
S U Z U K I ~ AWl) Y U K I M A S A H A Y A S H I
Institute o[ Molecular Biology, Faculty o[ Science, Nagoya University, Chikusa-Ku, Nagoya (Japan) (Received F e b r u a r y 12th, I964)
SUMMARY
I. When Escherichia coli cells are incubated in a medium lacking magnesium, they lose most of their ribosomes without loss of viability, as reported by McCARTHY1. 2. Upon the addition of magnesium, the cells accumulate "nascent ribosomal RNA" in the particles which appear to correspond to those synthesized in the presence of chloramphenicol. 3. The nascent ribosomal RNA has a slightly different sedimentation coefficient from that of the mature ribosomal RNA. This is probably due to the difference in secondary structure between these two types of RNA, a difference which can be shown by the hyperchromicity of the nascent form upon heating.
INTRODUCTION
It is known that, when cells of Escherichia coli are incubated in a growth medium lacking magnesium, they lose most of their original ribosomes without loss of viability 1. Upon the addition of magnesium such "ribosome-free" E. coli gradually resume the synthesis of ribosomal particles before the initiation of normal growth. The synthesis of ribosomal particles and r-RNA, in the early phase of recovery from magnesium-starvation, has been investigated in the present study. It has been found that, immediately after magnesium replenishment, the "ribosome-free" cells accumulate nascent r-RNA in unusual particles without the synthesis of typical ribosomes for a long period. These particles appear to correspond to those synthesized in the presence of chloramphenicol. Since such cells have neither pre-formed nor newly synthesized, typical ribosomes, a tangible quantity of the nascent r-RNA can be isolated. Direct chemical analysis shows that this RNA has a ribosomal type of ratio for its component bases. However, it possesses a sedimentation coefficient which is slightly different from typical r-RNA and also it has less hyperchromicity upon heating. Abbreviation: r-RNA, ribosomal RNA. " P r e s e n t address: N a t i o n a l I n s t i t u t e of Genetics, M i s h i m a ( J a p a n ) .
Biochim. Biophys. Acta, 87 (1964) 6 1 o - 6 2 o
FORMATION OF RIBOSOMAL RNA
611
MATERIALS AND METHODS
Bacterial culture Usually one loopful of E. coli B(I-I) cells were inoculated into 50 ml of Trisglucose medium 2. After overnight incubation the cells were transferred to fresh medium of the same composition to give a bacterial concentration of 1. 5. lO s cells/ml. They were then reincubated over 3 generations. The cells were collected and washed twice with magnesium-free medium (or saline) and then suspended in Tris-glucose medium lacking magnesium. The magnesium starvation was performed by culturing the bacteria under powerful aeration for 20 h. At the end of this period, viable count did not change, and the ribosomal content of the cell was decreased to a minimum, as described by ]V[CCARTHY1. The medium for the restoration from magnesiumstarvation contained o.I % casamino acids in addition to the usual Tris-glucose medium. This medium will be designated as the "restoration medium" throughout this paper.
Incorporation o~ isotopes For the measurement of isotope-incorporation into the RNA and the protein of whole cells, the following procedure was employed. Of two flasks (each containing 20 ml of the restoration medium), one (A) received 5#C of ~1~C]uracil (6.5/~C/ mmole), the other (B) 3.7 #C of [14C]leucine (7.41/*C/mg), and magnesium-starved cells to give 4" los cells per millilitre. Samples of 2 ml each were taken out after an appropriate interval of time and added (A) to 2 ml of cold IO % trichloroacetic acid in an ice bath, or (B) to 2 ml of hot trichloroacetic acid at 90° in a water bath in which case they were cooled after 15 rain. The precipitate was collected on a ultrafilter 984H (Hurlbut Paper Co. (U.S.A.)), washed with 5 % trichloroacetic acid, and then dried. The radioactivity was measured in a Geiger counter. For the preparation of the sample for sedimentation-analysis, El~C]uracil (o.I ~C) was added to IOO ml of medium at the outset of incubation.
Preparation o~ crude cell-extract Harvested cells were washed with o.oi M Tris buffer (pH 7.8) containing o.oooi M magnesium acetate, centrifuged and the pellet was then frozen immediately at --20 °. The frozen cells were ground with fine quartz sand for 2-3 rain and extracted with about 3 volumes of the above buffer. Sand and the cell debris were removed b y centrifugation at 7500 rev./min for IO rain. Deoxyribonuclease (EC 3.1.4.5) (DNAase, twice crystallized and obtained from Worthington Biochemical Corp., N.J. (U.S.A.)) was added, at a concentration of 5/~g/ml, at this stage.
Preparation o~ RNA The procedure employed here was essentially the same as that described previously 3. Bacterial cells were ground with quartz sand in the presence of sodium lauryl sulphate and extracted with 0.005 M Tris buffer (plK 7.8) containing 0.2 % sodium lauryl sulphate. The extract was shaken for 3 rain with an equal volume of 9 ° % (w/w) phenol in the cold. The aqueous layer was taken out, and another volume of Tris-sodium lauryl sulphate was added to the phenolic phase. The extraction was Biochim. Biophys. Acta, 87 (1964) 61o-62o
012
H. SUZUKI AND Y. HAYASHI
repeated. From the combined aqueous layer, the nucleic acids were precipitated with 2 volumes of cold ethanol, redissolved in o.o2 M acetate buffer - o . I M NaC1 (pH 5.o), and dialyzed against the same buffer for about 2 h. They were finally treated with DNAase (4 #g/ml).
Sucrose density-gradient centrifugation The crude cell extract was layered on to 27 ml of sucrose (5 % to 20 O//ogradient) containing the same Tris buffer as for the preparation of the cell extract, and was centrifuged using a Spinco L SW 25.1 rotor at 23 ooo or 25 ooo rev./min. For the fractionation of RNA, a gradient of 2.5 % to I5 % of sucrose 4 in 0,05 M acetateo.I M NaC1 (pH 5.0) was used. This centrifugation was performed using the same rotor at 25 ooo rev./min for 13 h. Io-15 drops were collected by dripping from the bottom of the tube. After the measurement of 1260rn/*, RNA was precipitated with cold 8 % trichloroacetic acid together with carrier protein. The precipitate was centrifuged, washed twice with 5 o//otrichloroacetic acid, dissolved in dilute ammonia solution, and then dried on a steel planehet. The radioactivity was measured either in a Geiger counter, or a gas-flow counter. In order to isolate separately either the I6-(I7) or the 23-(22.5) -S RNA component, the RNA preparation was subjected to the sucrose density-gradient centrifugation as described above. Two volumes of ethanol were added to precipitate the R N A and the precipitate was stored at --20 ° until use.
Nucleotide composition and chemical analysis Nucleotide composition was analyzed by the Dowex-i column chromatographic method as described b y OSAWA et al. ~. RNA was assayed by the orcinol reaction and protein by LOWRY'S method.
A bsorbancy-temperature profile The hyperchromicity of RNA at 260 m# was determined at different temperatures in 0.o05 M phosphate buffer (pit 6.3) containing I M NaC1 and using a thermostatically controlled spectrophotometer (Hitachi EPU). RESULTS
Disappearance of ribosomes in magnesium-starved cells The magnesium-starved cells lost practically all of the ribosomes of the types 30 S and 50 S (Fig. I), and contained a component with sedimentation coefficient of about 25 S. In order to estimate the amount of typical ribosomal particles which might still remain, the exponentially growing cells were labelled with 32P1 (o.oi mC/ml of culture medium). The cells were then washed and incubated in the absence of magnesium. After incubation, an extract was made from the cells in o.oooi M magnesium acetate and it was analyzed b y sucrose density-gradient centrifugation. From this experiment, the ribosomal content was estimated to be about 1. 3 % of the normally grown cells as judged b y the ~*P content in the 5o-S ribosome region. Larger particles (7° S and IOO S) were not observed in a sample derived from the medium containing a higher concentration (o.oi M ) o f magnesium.
Biochim. Biophys. Acta, 87 (1964) 61o-62o
FORMATION OF RIBOSOMAL R N A 50S
1.5
613
30S
,::L
1.c (D cD o,J
~ o.5
J
I
I
1o 2o Tube number
30
-,,5--
30
1.5
:m 1.0 E Q (0 o4
0.5 13 ..13
.......
Tube
26
I
number
Fig. i. Absence of ribosomes in crude extract from magnesium-starved cells, a, exponentially growing cells; b, magnesium-starved cells. Sucrose density-gradient, 5 % to 20 % in o.oooi M magnesium acetate-o.oi M Tris buffer (pH 7.8). Centrifugation for IO h at 23 ooo rev./min. Total amount of extract from approx. 3" 1°1° cells was applied.
Nucleic acids were prepared by the "phenol" method from magnesium-starved cells and these were subjected to sucrose density-gradient centrifugation. The bulk of A~60m/z absorbing material was detected in 4-1o S with only a trace of it in the 23-S region (Fig. 2b), where the typical r-RNA should reside (Fig. 2a). The results described in this section are in essential agreement with those reported by ]V[CCARTHY1.
Growth, and synthesis o/ RNA and o/ protein during the recovery /rom magnesiumstarvation A~50 m~ of t h e c u l t u r e in t h e r e s to r a t io n m e d i u m r e m a i n e d c o n s t a n t for m o r e t h a n 2 h. A t least 3 h were r e q u i r e d u n ti l the cells r e s u m e d a l o g a r i t h m i c growth. T h e first t race of ty p i c a l 3o-S a n d 5o-S ribosomes usually a p p e a r e d 2-3 h after t h e a d d i t i o n of m a g n e s i u m .
Biochim. Biophys. Acta, 87 (1964) 6Io-62o
614
H. SUZUKI AND Y. HAYASHI 23S
b
Q~
& 3_
~-2.0 O
E
o e~ 0.4
>~
1.o
4
<
I
10
o o.~
q
L
2~D 30 40 Tube number
10 20 30 Tube n u m b e r
40
Fig. 2. Absence of typical r-RNA in magnesium-starved cells, a, exponentially growing cells; b, magnesium-starved cells. Sucrose density-gradient, 2.5 % to 15 % in o.o5M magnesium acetate-o.I M NaCI (pH 5.0). Centrifugation at 25 ooo rev./min for 13 h.
RNA 3o0
" •20C 10c
./o
....... .P
~ - o - , ~
-'~-
30
....... T-Oo 90
,
120
Time (rain)
i
& c_ E
.c E
1000
_~ 10000
o
c
< z rr
o.
5000
5OO co
~6
~5
2 60 120 Time ( r a i n )
180
Fig. 3. Protein and RNA synthesis during the course of recovery from magnesium-starvation. a, chemical measurements; b, incorporation of isotopes.
Biochim. Biophys. Acla, 87 (1964) 6Io-62o
FORMATION O F RIBOSOMAL
RNA
6i 5
As shown in Figs. 3a and 3b, the synthesis of RNA as measured either by direct chemical determination or by [14C]uracilincorporation into RNA, started immediately after the addition of magnesium. On the other hand the synthesis of protein was strongly inhibited for about 3 h, as seen by the chemical analyses and by the incorporation of [~dC]leucine into protein. The synthesis then gradually recovered its normal rate.
Properties o/ the particles containing RNA synthesized in the early phase o/magnesiumreplenishment Crude extracts from the cells exposed to [14C]uracil for different periods of time were analysed by sucrose density-gradient centrifugation. With exposure for IO rain, most of the activity was detected as 24-25-S and I7-I8-S particles (Fig. 4a). The prolonged incubation (for 9 ° rain and 18o rain) in the restoration medium allowed these particles to accumulate in a considerable amount (Figs. 4b and 4c). The radioactivity peaks coincided with those manifested by A~e0 m#. These unusual particles 50S
30S
,.~ 0.8
~" 0 0 0 "E" ~:
P
i 0.6
30S
I
I
b
i
' _~o0o~-
~0
0 0.4
50S
E %-.
0.4
O4
8
L%
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C
~0.2 o
o
~ ~' N o.2
lOOO ~>,
t_2/ v
i
.13
/
.<[ o
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rY n.-
3b
2o 3'o Tube numbeP
o
Tube number
5is
.i s
c
0.6
80O0
T
0
10
20 30 Tube number
40
Fig. 4. S e d i m e n t a t i o n - a n a l y s e s of c r u d e e x t r a c t labelled w i t h [14C]uracil. E x p o s e d to [14C]uracil (o.oi #C) for: a, io m i n ; b, 90 rain; c, i 8 o m i n . Sucrose d e n s i t y - g r a d i e n t , t h e s a m e as in Fig. I. C e n t r i f u g a t i o n a t 25 ooo r e v . / m i n for: a, 9 h; b, c 7 h. A,80m v of 3o-S a n d 5o-S p e a k s are d u e to n o r m a l r i b o s o m e s a d d e d as reference.
Biochim. Biophys. Acta, 87 (1964) 6:to-62o
010
H. SUZUKI AND Y. HAYASHI
could be observed over a long period of time, and they were still present after the cells had started to grow exponentially. From the distribution of radioactivity in Figs. 4b and 4c, it is apparent that a considerable amount of 4-S RNA (probably soluble RNA) was also synthesized besides the particles mentioned above. Chloramphenicol is known to inhibit the synthesis of protein, and it accumulates "chloramphenicol-particles" (18 S and 25 S) when added to the cells which have been grown normallyd, 7. In the case of magnesium-replenishment also, the protein synthesis is strongly suppressed, together with the accumulation of the unusual particles. We therefore compared the sedimentation pattern of our unusual particles with that of chloramphenicol particles by the use of the analytical ultracentrifuge. For the preparation of chloramphenicol-inhibited cells, exponentially growing E. coli cells were incubated for 1.5 h with 200 ug of chloramphicol. The collected cells were used for the preparation of crude extract. Typical chloramphenicol-particles with sedimentation coefficients of 19 S and 15 S (uncorrected) could be seen in Fig. 5a in b
7_0
Chlor'amphenicol
tD
O~
a Mg2+ replenished
O
'
/ Fig. 5. Sedimentation-analyses of crude extracts from chloramphenicol-inhibited cells and Iron1 magnesium-replenished ceils. a, chloramphenicol-inhibited cells; b, magnesium-replenished cells. Photographs taken 16 rain after reaching 5° 74° rev./min. Solvent: o.oooi M magnesium acetate-o.I M Tris buffer (pH 7.8). the case of chloramphenicol-inhibited cells. The pattern of magnesium-replenished cell-extract is shown in Fig. 5b and is essentially the same as that of Fig. 5a, except t h a t no 50 S and 3o S peaks were seen in the latter.
Properties o/ the RNA synthesized in the early phase o/ magnesium-replenishment Magnesium-starved cells were labelled with [14Cluracil in the restoration medium for IO rain. They were mixed with an approximately equal amount of nnlabelled normal cells as the source of standard I6-S and 23-S r-RNA. RNA was then prepared by the "phenol" method and centrifuged in a sucrose density-gradient. The labelled RNA appeared in the 23-S and the I6-S regions and some in the 4-S region (Fig. 6). As deduced from the pattern of the crude extract (Fig. 4a), the radioactive RNA in the I6-S-23-S region was probably derived from the I7-S-25-S particles. The RNA labelled '16 S' showed a tendency to go ahead of the standard 16 S, and the
Biochim. Biophys. Acta, 87 (1964) 61o-6zo
FORMATION OF RIBOSOMAL
RNA
617
23S 3OO
0.~
T
/ 200
OA
= E
03
0.1
ki 100 "o~ 0
r~
b
10
2i0 Tube number"
30
Fig. 6. S e d i m e n t a t i o n - a n a l y s e s of R N A labelled w i t h [14C]uracil. E x p o s e d to [z4C]uracil for IO m i n . Sucrose d e n s i t y - g r a d i e n t , t h e s a m e as in Fig. 2. C e n t r i f u g a t i o n at 25 ooo r e v . / m i n for 13 h.
'23 S' behind the 23 S. This pattern is similar to the case of nascent r-RNA reported by ~/IITSUI et al. ~. Since the RNA was accumulated in the I7-S-25-S particles for a long period without the formation of typical 3o-S and 5o-S particles, and since no pre-formed particles (therefore no I6-S and 23-S r-RNA) were present in the cells, the r-RNA which was newly synthesized upon magnesium-replenishment could be obtained in quantity, which is directly observable by A260m# measurement. Magnesium-starved cells were incubated in the restoration medium for 9 ° rain without isotope, and the RNA was prepared by the "phenol" method. The preparation was mixed with a trace amount of s2P-labelled authentic I6-S and 23-S r-RNA, and centrifuged in a sucrose density gradient. The obtained pattern indicated just the reverse situation between absorbancy and radioactivity as compared with the preceding 14C-experiment (Fig. 7). The Az60m/~ of the newly formed r-RNA was slightly behind that of 23S
16S
~
I0OO "2
cD 0.4
O4
'4 o
o.~ ,
c
~
500
'~,
lb
~ Tube number"
:~-
30
Fig. 7. S e d i m e n t a t i o n - a n a l y s i s of R N A prepared f r o m cells i n c u b a t e d for 90 rain after the a d d i t i o n of m a g n e s i u m . Sucrose d e n s i t y - g r a d i e n t , t h e s a m e as in Fig. 2. C e n t r i f u g a t i o n at 25 ooo r e v . / m i n . A t r a c e of s2P-labelled a u t h e n t i c r - R N A f r o m n o r m a l l y g r o w i n g cells w a s added as t h e reference.
Biochim. Biophys. Acta, 87 (1964) 6 1 o - 6 2 o
0I~
H. SUZUKI AND Y. HAYASHI
[a2P]r-RNA in the I6-S region, and slightly ahead in the 23-S region. From this experiment, we could conclude that the slight difference in sedimentation coefficients noted in the l~C-experiment between the nascent r-RNA and the authentic r-RNA was a reality; and that it was not occasioned by an aggregation of smaller components with some of the r-RNA molecules. The nascent r-RNA in question was estimated to have sedimentation coefficients of both 17 S and 22.5 S, in accordance with those of pulse-labelled nascent r-RNA under the 'shift-up' condition reported by MIa'SuI et al. a.
The results of the base-ratio analyses on tile nascent r-RNA and the authentic r-RNA are presented in Table I. From the results, it is clear that the nascent r-RNA synthesized in the early stage of recovery from magnesium-starvation bore tile ribosomal type base-ratio. TABLE I NUCLEOTIDE COMPOSITION OF 'NASCENT RIBOSOMAL RN.X_' FROM MAGNESIUM-REPLENISHED CELLS Total R N A was p r e p a r e d from cells 12o rain after the addition of magnesium. ' N a s c e n t ribosomal R N A ' was t h e n separated f r o m low molecular weight R N A b y precipitation in 2 M NaC1. Sedi m e n t a t i o n - a n a l y s i s of the R N A so p r e p a r e d indicated t h a t it is m a i n l y composed of I7-S and 22.5-S RNA.
' N a s c e n t ribosomal R N A ' Ribosomal RNA
.4 denylic acid
Guanylic acid
Cytidylic acid
Uridylic acid
24. 4 24.i
31,4 31.9
21.i 21. 5
23.o 22.8
The data of hyperchromicity upon heating of the newly formed I7-S and 22.5-S r-RNA are shown in Figs. 8a and 8b, in comparison with those of authentic I6-S and 23-S r-RNA. All the RNA components used were isolated by sucrose densitygradient centrifugation from total RNA preparation. Each sample so prepared gave a single peak on sedimentation analysis, with a slight shoulder of the other material. Both 17 S and 22.5 S, especially 22.5 S, revealedhyperchromic effect to a~considerably lower extent than authentic I6-S and 23-S r-RNA respectively. DISCUSSION
MCCARTHY1 reported that, when E. coli cells are subjected to magnesium-starvation, they are depleted of preformed ribosomes. We have now found that, upon replenishment of magnesium ions to such cells, tile synthesis of what appeared to be the "nascent" or "premature" r-RNA (17 S and 22.5 S) having a ribosomal type of base-ratio is immediately resumed, while the synthesis of protein is suppressed for a long period. The nascent r:RNA is incorporated into particles which appear to correspond to chloramphenicol-particles 6& The situation therefore parallels that prevailing when bacteria are treated with chloramphenicol. A slight difference between the sedimentation coefficients of nascent and authentic r-RNA might be caused by the difference in their chain length. It is probable that the nascent r-RNA has the size corresponding to the mature r-RNA already in their incipient synthetic stage, and the difference in sedimentation coefficients is due to the difference in secondary structure. Although the evidence is not yet conclusive Biochim.
Biophys,
Acla,
87 (1964) 61o-62o
FORMATION OF RIBOSOMAL RNA
619
/,,~/,
/o..~_-9 16-S RNA 17-S RNA
20
C
o .XY'~ )
30
40
50
I
I
60
70
I
80
Temperature
b 2c
,o/
/
~
22.5-S RNA
b K
020 30
40 510 6() "ZO dO Tem perature Fig. 8. U l t r a v i o l e t absorbancy-temperature profile of mature and nascent r - R N A , a, I6-S r - R N A a n d I7-S n a s c e n t r - R N A ; b, 23-S r - R N A and 22.5-S n a s c e n t r-RNA.
on this point, both I7-S and 22.5-S r-RNA exhibited a significantly lower hyperchromicity upon heating than I6-S and 23-S r-RNA, thus suggesting that the nascent r-~RNA has less secondary structure than the I6-S and 23-S r-RNA. Nascent r-RNA detected b y pulse-labelling of the 'shift-up' cells also has sedimentation coefficients of 17 S and 22 S (see ref. 3). It forms a rapidly sedimenting aggregate much more readily than r-RNA at higher concentrations of magnesium s, which also suggests a difference in the secondary structure. NOTE ADDED IN PROOF
Experiments were performedrecently on the sedimentation behaviour of the nascent and mature r-RNA at near infinite dilution. Peaks were identified b y SH and 14C labels in a Tri-carb Liquid Scintillation Spectrometer. Tile behaviour of the minor component (16-17 S) aof 3H-labelled nascent r-RNA was, as shown in the present paper, definitely different from that of the mature one labelled with 14C. However, no difference was detected on the major component of nascent and mature r-RNA under the same conditions. Received August 7th, 1964 ACKNOWLEDGEMENTS
The authors wish to thank Dr. S. OSAWAfor much help throughout this study. Some of the experiments were performed at the Research Institute for Nuclear Medicine Biochim. Biophys. Acta, 87 (1964) 61o-62o
620
H. SUZUKI AND Y. HAYASHI
and Biology, Hiroshima University, where Professor A. SIBATANI gave us much fruitful advice. The work was supported by grants from the U.S. Public Health Service to Dr. OSAWA (GM lO466-Ol ), the Asahi Press and the Ministry of Education of Japan. REFERENCES 1 g . J. MCCARTHY, Biochim. Biophys. Acla, 55 (1962) 88o. A. ISHIHAMA, N. MIZUNO, M. TAKAI, E. OTAKA AND S. OSAWA,J. l~/Iol. BioL, 5 (1962) 251. 3 H. MITSUI, A. ISHIHAMA AND S. OSAWA, Biochim. Biophys. Acta, 76 (1963) 4Ol. * M. HAYASHI AND S. SPIEGELMAN,Proc. Natl. Acad. Sci., U.S., 47 (1961) 1564. 5 S. OSAWA, •. TAKATA AND Y. HOTTA, Biochim. Biophys. Acta, 28 (1958) 271. * M. NOMURA AND J. D. WATSON, J. Mol. Biol., i (1959) 2o4. C. G. KURLAND, M. NOMURA AND J. D. WATSON', J. Mol. Biol., 4 (1962) 388. 8 S. OSAWA, Biochim. Biophys. Acta, 42 (196o) 244.
Biochim. Biophys. Acta, 87 (1964) 61o-62o