Biogenesis of ribosomes: Free ribosomal protein pools in Escherichia coli

J. Nol.

Biol. (1972) 69, 279-301

Biogenesis of Ribosomes : Free Ribosomal in Escherichia coli

Protein Pools

R. S. GUPTA AND U. N. SINGH Molecular Biology Unit Tata Institute of Pundamentul Research Bombay-5, India (Received 1 December

1971, and in revisedform 28 March

1972)

Proteins from ribosomal subunits (30 s and 50 S) have been fractionated into split (SP-30 and SP-50) and core (core-30 and core-50) proteins. Antisera prepared in rabbit against them are shown to be highly group-specific as judged by the Ouchterlony (1967) double diffusion test and by precipitin reaction in solution. Various parameters which influence the immuno-precipitation of these proteins by specific antisera have been investigated. It is demonstrated that under controlled conditions this provides a sensitive and reliable method for characterization and quantitative estimation of free ribosomal proteins. The technique has been successfully applied in the investigations of various properties of free ribosomal protein pools existing in Escherichia co& It is concluded that free ribosomal proteins in E. coli may constitute 8 to 14% of total soluble proteins under different growth conditions. Relative pool sizes of four classes of proteins expressed as a percentage of the total soluble proteins in bacteria grown in L broth (doubling period, 30 min) are estimated to be core-30, 2.1; core-50, 3.4; SP-30, 3.6; SP-50, 5.0. From the studies on bacteria grown in different media (doubling period, 30,42 and 60 min), we further conclude that the amount of free proteins increases with the growth rate so as to constitute a constant fraction (7 t,o 9%) of total ribosomal proteins. Relative pool-sizes corresponding to four classes of ribosomal proteins, however, remain unaltered by different growth rate. Studies on the time-course of incorporation of labelled amino acids show that the level of radioactivity (specific activity) in free ribosomal protein pools reaches a saturation value in 2 to 4 minutes with an average half-life of about 50 seoonds. Kinetic curves (specific activity versus time) for free and bound ribosomal proteins are found to be compatible with a precursor-product relationship. Pulseand-chase experiments further suggested that there is no significant breakdown of proteins in pools and that they are quantitatively transferred to mature ribosomes. Relative pool-sizes and kinetic behaviour of free ribosomal proteins indicate that these proteins are synthesized in a co-ordinate manner. Various possible mechanisms for a co-ordinate synthesis of ribosomal RNA and proteins at the level of subunits and at the level of whole ribosomes are discussed.

1. Introduction In vivo assembly of ribosomespresents a unique problem, as it involves co-ordination between the syntheses of two distinct classesof macromolecules, RNA and proteins, and an orderly interaction betweenthem. The regulation of synthetic processes may be considered to operate at two different levels: (i) synthesis of ribosomal RNA 279

280

R.

S. GUPTA

AND

U.

N.

SINGH

and proteins at the level of subunits; and (ii) transcription of two major ribosomal RNA’s (23 s and 16 s), which are found to be present in equimolar amounts in exponentially growing bacteria. Recent studies (Traub & Nomura, 1968; Nomura & Erdmann, 1970) on the reconstitution of ribosomal subunits in vitro have clearly indicated that the structural information contained in various macromolecular components is adequate for the assembly of biologically active particles. These studies have further demonstrated that the interaction between RNA’s and proteins is not a random process and that the assembly proceeds through a series of sequential steps involving intermediate sub-ribosomal particles (Mizushima & Nomura, 1970; Schaup, Green & Kurland, 1970 ; Traub & Nomura, 1969). The existence of such intermediates in the biogenesis of ribosomes has been recognized for some time. Roberts, Britten & McCarthy (1963) presented kinetic evidence in support of ribosomal precursors in exponentially growing bacteria. Mangiarotti, Apirion, Schlessinger & Silengo (1968) and Osawa, Otaka, Itoh $ Fukui (1969) have observed particles sedimenting at 30 to 32 s and 40 to 43 s which they implicated in the maturation of the 50 s ribosomal subunit. Accumulation of ribosomal precursors has been reported under a variety of experimental conditions (MacDonald, Turnock & Forchhammer, 1967 ; Lewandowski & Brownstein, 1969; Guthrie, Nashimoto & Nomura, 1969; Hosokawa & Nomura, 1965). Much of the work on the biogenesis of ribosomes in recent years has centred around sub-ribosomal particles, and a large extent has depended on their separation and characterization by sedimentation velocity on sucrose density gradients. Several workers (Cozzone, Marvaldi & Marchis-Mouren, 1969; Gierer & Gierer, 1968; Sells & Davis, 1970; Young & Nakada, 1970; Santer et al., 1968) have inferred the existence of considerable amounts of ribosomal precursor pools in bacteria. Due to the lack of appropriate techniques, much of the information available on various properties of the precursor pools in bacteria is derived from indirect observations and is highly fragmentary. Such information is of course crucial to our understanding of the regulatory mechanism operating at the synthetic level in the biogenesis of ribosomes. In this paper we describe the quantitative aspects of the immuno-precipitin reaction given by ribosomal proteins against specific antisera. The technique is applied in the study of the dynamic behaviour of free ribosomal protein pools in E. coli. Systematic differences observed in pool sizes of split and core proteins of 30 s and 50 s ribosomal subunits indicate a co-ordinate synthesis of these proteins in exponentially growing bacteria. Isotopic incorporation studies have provided support for a precursor role of free protein pools in the assembly of ribosomal particles. Various possible mechanisms of co-ordination of the synthesis of two ribosomal subunits are examined in the light of available information.

2. Materials (a)

and Methods Chemicals

lW-Labelled ChZoreZEa protein hydrolysate (300 pCi/mg) was obtained from the Bhabha Atomic Research Centre, Bombay, India. Complete Freund’s adjuvant was obtained from D&o Laboratories, Detroit, U.S.A. All other chemicals were reagent grade and are readily available from commercial suppliers. (b)

Bacteria

and

culture

conditions

E. co.% strain KlO wild-type was used throughout these studies. Bacteria were grown in a shaker at 37°C. For labelling experiments, minimal medium M9 (Roberts, Abelson, Cowie, Rolton & Britten, 1957) supplemented with 0.2% glucose was used. L broth (Luria & Burrows, 1957) was used as complete medium for large-scale ribosomal preparations.

BIOGENESIS (c)

OF

of riboscnnes

Preparation

RIBOSOMES

IN

and isolation

E.

COLI

of ribosowd

281

subunita

Bacteria were harvested at late logarithmic phase, from 10 1. of culture growing in L broth, by centrifugation at 7000 g for 6 to 7 mm. The bacterial paste was washed once with standard high Mga+ buffer (0.01 M-Tris.HCl (pH 7.5), 0.05 M-KCl, 0.005 M-CzHeSH, 0.01 M-UuLgneSiU~ acetate) at 4°C. Washed cells were resuspended in 80 ml. of standard high Mg a+ buffer and an extract was made in a French pressure cell. The extract was cleared of cell debris and membranous material by centrifugation at 30,000 g for 30 min. Ribosomes were obtained by centrifuging the 30,000 g supernatant fraction at 105,000 g for 25 hr. The pellet was suspended in 80 ml. of 5% sucrose (in standard high Mga + buffer) and centrifuged again at 105,000 g for 2.5 hr to free it from attached proteins. Washed ribosomes were suspended in standard low magnesium buffer (0.01 r.r-Tris. HCl (pH 7*5), O-006 M-CzH,SH, 0.05 ~-Kc1 and 0.0002 M-magnesium acetate) and dialysed in the cold at 4°C for 8 hr against 5 1. of the same buffer. The dialysed solution containing dissociated subunits was cleared of any aggregate by low-speed centrifugation at 12,000 g for 10 min. Separation and isolation of ribosomal subunits were carried out essentially by the method described by Tissieres, Watson, Schlessinger & Hollingworth (1959). The ribosomal subunit preparations obtained after 4 cycles of resuspension and centrifugation contained less than 3% of the other subunit, as judged by sucrose density-gradient analysis. Ribosomal subunits were then finally suspended in a small volume of standard low Mg2+ buffer to give a concentration of about 5 mg/ml.

(d)

Preparation

of core and split

proteins

Core and split proteins were prepared according to the method described by Itoh, Gtaka & Osawa (1968). To a 5-ml. suspension of the ribosomal subunit an equal vol. of a cold solution containing 2.0 M-Lick, 0.2 M-MgCl,, 0.2 M-Tris. HCl (pH 7.5) was added and after gentle mixing it was allowed to stand for 12 hr at 4°C. The mixture was centrifuged at 105,000 g for 6 hr. The top 7 to 8 ml. containing split proteins were carefully pipetted out and preserved and the lower 1 to 2 ml. were discarded. The pellet was rinsed once with standard low Mg”+ buffer and re-suspended in 5 to 6 ml. of the same buffer. To the pellet suspension (which contains core particles) an equal vol. of a cold solution containing 8 Murea and 4 M-LiCl was added (Spitnik-Elson, 1965). After keeping it overnight in the cold, the RNA was removed by centrifugation. The supernatant fraction contained core proteins. Both core and split proteins were dialysed against 5 1. of standard low Mgaf buffer with one change. Amounts of proteins were estimated by Lowry’s method (Lowry, Rosebrough, Farr & Randall, 1951).

(e)

Preparation

of antisera

Rabbits were immunized four times at weekly intervals against various ribosomal protein preparations mixed with an equal vol. of Freund’s adjuvant. Animals were bled 15 days after the administration of the last dose and antisera were tested for reactivity against total ribosomal proteins as well as against specific classes of proteins. Antisera obtained from repeated bleedings of the animals at appropriate time-intervals were pooled and stored at -20°C in the presence of 0.1 ye sodium azide added as preservative. Group specificities of these antisera were tested by the qualitative Ouchterlony doublediffusion technique (Ouchterlony, 1967). The plates were routinely developed in the cold at 4°C; this was found to give sharper precipitin bands than those obtained at room temperature. For quantitative immuno-precipitation, antigen preparations were cleared of any aggregated particles by centrifugation at 12,000 g for 10 min. To varying amounts of antigen preparations, 0.2 ml. of specific antiserum was added. The final volume in each After mixing, the tubes were case was made up to 0.5 ml. with standard low Mg ‘+ buffer. kept in the cold for 6 hr and then centrifuged at 7000 g for 10 min. The immuno-precipitate was washed 3 times with 1 ml. of cold saline (O*85°/0 NaCl) by centrifugation and dissolved in 0.5 ml. of 0.1 N-NaOH. Fractions of this solution were used for estimating proteins or for measurement of radioactivity in a liquid-scintillation counter.

R.

282 (f)

S. GUPTA

Labelling

AND

U.

N.

of cells in kinetic

SINGH experiments

Bacteria were grown in 50 ml. of MS medium at 37°C. When the cell density was approximately 2 to 5 x lo* cells per ml., 100 PCi of 14C-labelled Chlorella protein hydrolysate was added. &ml. samples were removed at appropriate time intervals and collected in cooled tubes which were kept immersed in liquid nitrogen to prevent further incorporation. In pulse-and-chase experiments, cells (in 50 ml. culture) were pulse-labolled with 100 PCi of 14C-labelled Chlorella protein hydrolysate for 30 sec. The label was then diluted by adding Casamino acids at a final concentration of 1% at zero time. Samples were removed as before at different time-intervals. Cells were washed twice with standard high Mg”+ buffer. About 5 x lOlo cells growing under identical conditions were added to each sample as carriers. Cells were kept frozen at -50°C until they were processed. Cell extracts from frozen samples were prepared according to the method described by Godson (1967). After removing cellular debris by low-speed centrifugation, the extracts were incubated in the presence of puromycin (50 pg/ml.) at 37°C for 10 min to release nascent peptide chains from the ribosomes. Heavier particulate materials were sedimented by centrifugation at 30,000 g for 30 min. The supernatant fraction was dialysed against 2 1. for 4 hr. Washed ribosomes were prepared as described in of standard high Mg2+ buffer section (c) above and suspended in 3 ml. of standard low Mg2+ buffer containing 4 M-urea and 2 r,r-LiCl. After standing overnight at 4”C, the precipitated RNA was removed and the solution was dialysed against 11. of standard low Mg ‘+ buffer. Spociflc activities of different classes of proteins in the dialysed solution (freed from any aggregated particles by lowspeed centrifugation) were determined by precipitation against group-specific antisera. The supernatant fraction obtained after the first centrifugation at 105,000 g in the preparation of ribosomes was further cent,rifuged at the same speed for 6 hr to remove any subunits and ribosomal precursor particles. The upper two-thirds was carefully pipetted out and designated as soluble extract. This was used to determine specific activities of free ribosomal proteins by precipitation against group-specific ant,isera.

(g) Measurement

Aqueous samplesof scintillation

counter.

14C-labelled 5 ml. of Bray’s

material scintillation

of radioactivity were fluid

counted (Bray,

in a Packard Tricarb liquid1960) were added to up to 0.5

ml. of aqueoussolution.

3. Results (a) SpecQicities

of, and quantitative four

&l&W

immune-precipitation Of ribosoma~ protein8

by antisera

directed

against

Since most of the work described in this paper depended on the reliability of the specific immuno-precipitin reaction in the quantitation of various parameters, the antigen-antibody systems were investigated in detail. It is demonstrated that the antisera used in these studies were highly group-specific with no detectable crosscontamination, by the Ouchterlony double-diffusion test (see Plates I and II). It is clearly seenthat antisera directed against 30 s split and core proteins show precipitin bands only against total 30 s proteins and give no reaction against 50 s proteins. On the other hand, antisera against 50 s split and core proteins react only with homologous preparations derived from 50 s particles. Specificities of the four classes of antisera were further tested against purified split and core proteins derived from 30 s and 50 s ribosomal subunits. Antisera in these tests showed precipitin bands only against homologous antigenic preparations. Normal serum in all casesshowed no reactivity against any of the proteins. &me the meohanismof antigen-antibody interaction is still not clearly understood, the use of quantitative immuno-precipitation as an analytical tool must necessarily

BIOGENESIS

OF

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IN

E. COLI

253

be basedon empirical considerations. The simple formulation described below provides a useful means of evaluating the reliability of the technique. Consider a constant amount of specific antiserum to which varying amounts (A) of homologousantigen have been added. The amount (P) of precipitate is then given by :

(1) where j is the fraction of antigen precipitated and r the weight ratio of antigen to antibody in the precipitate. This is a very general expression in which both f and r are assumedto vary with antigen-antibody ratio in the reaction mixture. In our preliminary studies, we used antisera prepared against total ribosomal proteins. In Figure 1 the amount (P) of precipitate is plotted against that (A) of total ribosomal proteins, the amount of antiserum directed against total ribosomal proteins being kept constant. The curve shows a rather complex behaviour, which is presumably due to the heterogeneity of the antigenic determinants present in total ribosomal proteins. This could also be a refiection of wide variations in solubilities of different classesof ribosomal proteins. In Figure 2 the results of immuno-precipitation of four classesof ribosomal proteins (SP-30, SP-50, core-30 and core-50) by homologousantisera are presented.These curves were constructed by pooling data from four independent experiments. The precipitin curves (P verse A) under the present experimental conditions are approximately linear over a wide range of variations in concentrations of three classes of proteins, namely, core-30, SP-50 and core-50. The P versus A curve for 30 s split protein, however, shows complex behaviour. 000

600 X

s a D 2 ,o 1% 400P h .E i2

t

,YX’ dX-Yx

/

200-/ X

0 Antigen

added

(pg)

FIG. 1. Immune-precipitin curves obtained in reactions with antisera directed against total ribosomal proteins. In this experiment 0.2 ml. of antiserum preparation was mixed with varying amounts of total ribosomal proteins, the final volume (0.5 ml.) of the reaction mixture being kept constant. Precipitation was carried out in the cold as described in Materials and Methods, section (e). In this Figure, amounts of immune-precipitates are plotted against that of ribosomal proteins added to the reaction mixture. -X-X-, Against antisera; -- O--0 --, normal serum.

284

R. (a) Core

30

S. GUPTA ’

AND

U.

N.

SINGH 200

’

(b) Split

I

30 - 150

- 100

- 50

50

100

150

200

50

Antigen

added

100

150

200

(pg)

FIG. 2. Immuno-precipitation of split and core proteins obtained from 30 s and 50 s particles by homologous antisera. This experiment was essentially similar to that described in Fig 1. Antisera directed against the 4 classes of ribosomal proteins ((a) core-30, (b) core-50, (0) split-30 and (d) split-50) were treated separately with corresponding antigen preparations. Amounts of precipitate were quantitated as described in Materials and Methods, and are plotted against amounts of ribosomal proteins in the reaction mixture. The amount (0.2 ml.) of antiserum and the flnal volume (0.5 ml.) of the reaction mixture were kept constant. Points refer to four different experiments.

In kinetic studies on the time-course of incorporation of labelled amino acids into bacteria, we have inferred values of specific activities of free ribosomal protein pools from the observed specific activities of immuno-precipitates. The reliability of these estimates greatly depends on the values of r, i.e. the ratio of antigenic protein to antibody in the immuno-precipitate. To investigate this aspect, labelledribosomes were prepared from bacteria grown in the presence of 14C-labelled Chlorellu protein hydrolysate. Ribosomal subunits in this casewere isolated by sedimentation on a sucrose density gradient. Core and split proteins were obtained as described in Materials and Methods. It can be easily shown that the relationship between specific activity (S,) of immuno-precipitate and that (S,) of protein is given by:

Thus for a constant antigen-antibody ratio in the reaction mixture S, should vary linearly with S,, and r can be readily obtained from the slope of the straight line. A representative S, versus S, plot is shown in Figure 3. Table 1 summarizes values of r for different amounts of ribosomal proteins in the reaction mixture. The amount (0.2 ml.) of antisera was kept constant in all the experiments.

BIOGENESIS

OF

Specific

RIBOSOMES

activity

of the antigen

IN

E. COLT

285

(cts/min/~g)

FIG. 3. Composition of antigen-antibody complex obtained in the precipitin reactions described in Fig. 2. In this experiment, 14C-labelled ribosomal protein fractions of varying specific activities were treated with homologous antisera, keeping the antigen-antibody ratio in the reaation mixture constant. Specific activities of immuno-precipitates were determined 88 described in Materials and Methods, section (g) and are plotted against that of ribosomal proteins. Only one representative plot for core-60 protein is presented in this Figure. Essentially similer linear reletionships were observed for other protein fractions. Antigen-antibody ratios (r) estimated from slopes of linear plots (8, veraua 8,) are summarized.

Equation

(2) can also be written

as:

si = C/A 1 + Ir1 ’ ( where C is the amount of radioactive material in labelled proteins. Thus S, versus 1/A, where C and r remain constant, should give a straight line. This is essentially a rationalization of the isotope-dilution technique used in the estimation of free ribosomal protein pools in bacteria. As seen from Figure 4, Si varies linearly with l/A over a wide range of concentrations. These curves were used as standard curves in the isotope-dilution method for estimations of free ribosomal proteins in E. co&. (b) Estimation

of free ribosomal proteins in soluble extract

Plate III shows the immunological reaction of ribosome-free soluble extract from E. coli with specific antisera in the Ouchterlony double-diffusion test. Antisera directed against SP-30, SP-50 and total ribosomal proteins show strong precipitin bands. The bands corresponding to antisera against core proteins were always faint and appeared only after prolonged incubation. For quantitative estimation of free ribosomal protein pools in bacteria, cells grown in L broth were harvested in different phases of cell-growth. Ribosome-free cell extracts were prepared as described in Materials and Methods. Two methods were used in these studies : (i) quantitative immuno-precipitation; and (ii) isotope-dilution. In (i), 0.2 ml. of antiserum was incubated in the presence of different amounts of cell extracts under conditions identical to those used in standard precipitin reactions (Figs 2 and 4). Amounts of cell extracts were appropriately adjusted so as to obtain all the measurements on the linear part of the curves in

286

R.

S. GUPTA

AND

U.

TABLE

Compositions

(weight

N.

SINGH

1

ratio r) of immuno-precipitates obtained antibody ratio in the reaction mixtures

(Amount Class of proteins

Core-30 Split-30 Core-50 Split-50

for different

Antigen-antibody ratio in immune-precipitate of antigenic preparations in &/0.2 ml. of antisera mixture) 25 50 100 0.10

0.08 0.10 0.18 0.16

0.09 0.16 0.18

0.10 0.11 0.16 0.15

antigen-

in reaction

150 0.11 0.10 0.16 0.18

Values of antigen-antibody ratio in immune-precipitates for four classes of ribosomal proteins are summarized in this Table. Quantities of proteins were expressed in gram amounts. For a given antigen-antibody ratio in the reaction mixture, ratio T was estimated from the slope of a linear plot 8, ~.rsua 8, as described in the legend of Fig. 3. Note that compositions of immuno-precipitstes for all the four classes of ribosomal proteins remain unchanged over sixfold variations in the antigen-antibody ratio in reaction mixtures. Concentrations (in mg/ml.) of ribosomal protein preparations used in these studies were: core-50, 0.56; oore-30, 1.16; split-30, 0.62; split-SO, 0.50.

80

(a) Core-30

I

I

I

I

I

I

I

I

J 100

(b) Core - 50

- 75

60-

-50 -25

5

IO

15

20

25

IO

20

30

40

I 50 , 80

(d) Split-50

i/Antigen

x 1000Pg

‘t

Pm. 4. In this experiment a constant amount of kbelled antigenic preparation (ribosomel protein fraction) was added to varying amounts of corresponding u&belled proteins. Precipitation was carried out as described in Figs 1 and 2. In this Figure specific activities (8,) of immunoprecipitates are plotted sgeinst l/A, where A is the amount of ribosomal proteins in the reaction mixture, in accordance with equation (3). In each graph, -m-m-refers to 100 jd. of 14C-labelled core (or split) antigen; -X-Xrefers to 50 ~1. of 14C-labelled core (or split) antigen.

BIOGENESIS

OF

RIBOSOMES TABLE

IN

E. COLLI

281

2

Amounts of pee ribosomul proteins pools in soluble extracts of E. coli grown in L broth Ribosomal Class

of proteins

Core-30 Split-30 Core-60 Split-50

protein -total Immuno-precipitation technique 2.10&0.12 3.57&0.08 3.35f0.17 4.97kO.15

pools soluble

expressed as y0 of protein Isotope-dilution technique 2.18 3.55kO.15 3.54f0.37 515f0.02

Relative amounts of free ribosomal proteins present in soluble bacterial extract estimated by (i) immune-precipitation and (ii) isotope-dilution methods are presented. Experimental details are described in the text. Precipitations were carried out in triplicate for each sample. Figures in the Table refer to mean values obtained from measurements on 4 different preparations of soluble bacterial extract.

Figures 2 and 4. In the isotope-dilution experiments, the amounts and specific activities of labelled proteins were the same as those in Figure 4. Under these conditions, from the observed amounts and specific activities of immuno-precipitates, relative sizes of free ribosomal protein pools can be readily estimated. It may be pointed out that although in principle the two methods are not entirely independent of each other, together they provide an adequate check on the reliability of these estimates obtained from specific immuno-precipitation. Table 2 summarizes relative amounts of free ribosomal proteins in E. coli belonging to four different classes of ribosomal proteins. The values are expressed as percentages of total soluble proteins. Close correspondence between the values derived from two types of measurements added to our confidence in the reliability of these estimates. Although the isotope-dilution technique is definitely preferable to the quantitative immuno-precipitation, the latter is more convenient for routine analysis. Since the two methods provide equally reliable estimates, the quantitative immuno-precipitation was used in later experiments. It is apparent from these data that while relative sizes of various free ribosomal protein pools are of the same order of magnitude, there are a number of noticeable systematic differences between different classes. For example, the amount of free 50 s ribosomal proteins is always found to be greater than that of 30 s proteins, and for each subunit the amount of split proteins is greater than that of core proteins. This pattern has been consistently observed in all measurements and we do not consider it an artifact. Indeed, this may have important physiological significance in a co-ordinate synthesis of ribosomal proteins. (c) Variation of ribosonmlprotein pools under different growth conditions Several workers (Schaechter, Maalee & Kjeldgaard, 1958; Ecker & Schaechter, 1963; Kennel t Magasanik, 1962; Neidhardt, 1964) have observed that the ratio of the number of ribosomes to the total amount of proteins in exponentially growing bacteria is proportional to the growth rate constant. Schleif (1967) showed further that the relative rate of synthesis of ribosomal proteins as compared to that of total proteins is also proportional to the growth rate of the bacteria. These observations

288

R.

S. GUPTA

AND TABLE

Relative amounts of ribosomal

Growth medium

(iii)

Free Core-30 1.20 1.40 2.05

proteins

U.

SINGH

3

in free and bound form in E. coli grown in &fleered media

ribosomal proteins in soluble (O/c total soluble proteins) SP-30 Core-50 2.15 2.65 3.70

N.

1.55 2.55 3.15

extract SP-50

Bound ribosomal proteins (% of proteins in crude extract)

3.00 3.80 4.95

49 (20.7) 57 (29.2) 62 (40.5)

In these studies, the reletive amounts of free ribosomal proteins in solublo extrects from E. coli grown in 3 different media: (i) minim81 medium, (ii) minim81 medium supplemented with Cesamino acids (0.5%) and (iii) complete L broth were estimated by quantitative immunoprecipitation 8s described in the text. Doubling periods in (i), (ii) 8nd (iii) were estimated to be 60, 42 and 30 mm, respectively. Amounts of bound cellular ribosomal proteins were also determined in the s8me experiments by extensive centrifugation (2.5 hr at 105,000 g followed by further centrifugation for 6 hr at the s8me speed) of the crude extract (30,000 g supernatant). The values in brackets correspond to the proteins present in the sediment obtained after first centrifugation and 8re usually referred to 8s mature ribosomal proteins.

have led to numerous speculations regarding the role of free ribosomal proteins in the assembly of ribosomal particles in micro-organisms (Maalee, 1969). It was of considerable interest to know the corresponding variations in the size of free ribosomal protein pools with the growth rate. In these studies, E. coli were grown in three different media : (i) minimal M9 medium; (ii) minimal M9 medium containing 0.5% Casamino acids mixture and (iii) complete L broth; doubling periods in these media at 37°C were estimated to be 60, 42 and 30 minutes, respectively. The amounts of free ribosomal proteins (SP-30, SP-50, core-30 and core-50) were estimated as described previously (section (b) above). Results of these analyses are summarized in Table 3. Amounts of bound ribosomal proteins were also determined and the values are expressed as the percentage of proteins in crude cellular extract (30,000 g supernatant fraction). The amounts of bound ribosomal proteins obtained from these experiments are considerably higher than those reported by earlier workers (Maalee & Kjeldgaard, 1966; Neidhardt, 1964). This is primarily due to extensive centrifugation of the crude extract (25 hr at 105,000 g, followed by further centrifugation for 6 hr at the same speed)used in these studies. The estimates presented in Table 3 refer to the total amounts of proteins associatedwith the particulate fraction (70 s, 50 s and 30 s) including that in ribosomal precursor particles. The amounts of proteins present in the sediments obtained after first centrifugation (shown within brackets), usually referred to as mature ribosomal proteins, are comparable to the values reported in the literature. It is interesting to note that relative amounts of free ribosomal protein pools increasein parallel with the amounts present in the particulate fraction. (d) Kinetic studies of the synthesis of ribosomal as a precursor

proteins: free protein to that in mature ribosomes

in soluble extract

Results presented above have indicated the existence of considerable amounts of free ribosomal proteins in soluble bacterial extracts. These observations give little

BIOGENESIS

OF

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IN

E. COLI

289

information about the physiological role of these proteins. The possibility that such a large pool-size could be an artifact caused by an extensive breakdown of mature ribosomes during extraction cannot be excluded a priori. Kinetic studies on the incorporation of labelled amino acids into free (in soluble extracts) and bound (in ribosomes)ribosomal proteins presented in this section rule out any such possibility. The data provide support for our contention that ribosomal proteins in soluble extracts constitute a physiologically distinct compartOment,and that they serve as a precursor to proteins in mature ribosomes. (e) Time-course of incorporation of labelled amino acids into ribosomal proteins In these experiments, incorporation of labelled amino acids added in the form of 14C-labelled Chlorella protein hydrolysate into four classesof ribosomal proteins (SP-30, SP-50, core-30 and core-50) as defined earlier was followed. Specific activities of free and bound proteins in samplesremoved at various time-intervals were derived from the observed specific activities of immuno-precipitates obtained by reaction against four group-specific antisera. The reliability of these estimates is adequately supported by our observation (Fig. 3 and Table 1) that the composition of antigenantibody complex remains unchanged over a wide range of variations in the antigen-antibody ratio in the reaction mixture. As a further precaution, all precipitin reactions were carried out under identical conditions, keeping the same antigenantibody ratio. Figure 5 summarizes the results of a typical kinetic experiment in which specific act,ivities of various classesof ribosomal proteins both in the free and bound state are plotted against time. It showsthat radioactivity is rapidly incorporated into free ribosomal proteins with no detectable lag observed within the limits of our measure. ments. Specific activities of proteins in mature ribosomesare much lower than those of free proteins during the early period of incorporation. The curves in this case exhibit a noticeable lag. The distinctive features of the two sets of curves emphasized here exclude the possibility of cross-contamination between free and bound proteins to any significant extent. Further, their kinetic behaviour strongly suggestsa precursor-product relationship between them. We have attempted a quantitative evaluation of this aspect (seeAppendix). It may be pointed out that each of the four classesof ribosomal proteins as defined here is composedof several distinct molecular components. In spite of this chemical heterogeneity, the kinetic behaviour of split proteins of both 30 s and 50 s subunits in soluble extracts appearsto be relatively simple. As seenin Figure 5, the time-course of incorporation of radioactivity into the above two compartments follows simple first-order kinetics. This is compatible with a rapid equilibration of exogenous labelled amino acids with endogenousamino acids from the precursor pool. Specific activity versus time curves for core proteins in soluble extract, on the other hand, show a noticeable deviation from first-order kinetics. This is more particularly pronounced in the caseof core-30 proteins. Similar patterns have been consistently observed in a number of experiments and we believe that core proteins, as defined operationally in this work, consist of components which are kinetically more heterogeneousthan those in split proteins. (f) Pulse-chase experiments In these experiments, exponentially growing cells were pulse-labelled with 14Clabelled Chlorella protein hydrolysate (2 &X/ml.) for 30 seconds. The label in the

R.

S. GUPTA

iZSD

U.

N.

SIXGH

(b) Splbt-30

Id) Split-50

25

50

75

Time (min I

FIQ. 5. Time-course of incorporation of 14C-labellod amino acids into 4 different classes of ribosome1 proteins in soluble extracts and in mature ribosomes. In these studies 100 @i of W-labelled ChZoreZZa protein hydrolysate were added to an exponentially growing culture of E. coli at zero time. Samples were removed at different times and specific activities of four ribosomal protein fractions (SP-30, SP-50, core-30 and core-50) in soluble extracts as well as in mature ribosomes were determined as described in Materials and Methods. In this Figure, specific activities are plotted against time: (a) core-30; (b) split-30; (c) core-50; (d) split-50. --O-O-, Soluble extract; --@-a---, mature ribosomes.

medium was then diluted by adding Casamino acids mixture (final concn 1%) at zero time. Samples were removed at appropriate time intervals and specific activities of split and core proteins in both free and bound states were determined as described earlier. Results of a typical experiment plotted as specific activities versus time are shown in Figure 6. Exponential loss of radioactivity from free ribosomal protein pools in the soluble extract is consistent with first-order kinetics as observed in incorporation experiments (Fig. 5). Values of ra,te constants (0.5 min-l to 0.6 min-1) from the pulse-chase experiment tend to be lower than those (O-7 mine1 to 1.5 min-l) derived from incorporation curves shown in Figure 5. Besides the complex nature of these pools, which is quite obvious for core proteins, the assumptions implicit in the treatment of these data as first-order kinetics may also be partly responsible for this discrepancy. These limitations are examined in detail in the Appendix. In contrast to an exponential loss of radioactivity from free ribosomal protein pools, incorporation of amino acids into mature ribosomes continues for several minutes. On certain assumptions, continued incorporation of radioactive amino acids into ribosomes can be treated as first-order kinetics. Values of rate constant (0.4 min- l to 0.6 min-‘) estimated from these data are in excellent agreement with those derived from the observed loss of radioactivity from free ribosomal proteins. These observations strongly suggest that proteins in soluble extract are quantitatively transferred to mature ribosomes and that there is no significant breakdown of ribosomal proteins in exponentially growing bacteria.

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(bl S@f-30 S#-30

(dl Spld - 50

t

t 2.5

I

I 50

I

I 75

IO 0

I

I 25

I

I 50

I

I 75

I

Twne (min)

FIG. 6. Kinetic studies on the incorporation of lebelled amino acids into ribosomal proteins; pulse-and-chase experiment. In this experiment cells in an exponentially growing oulture were pulse-labelled for 30 set with ‘*C-labelled amino acids (Chlorella protein hydrolysete) followed by addition of Cassmino ctaids (1%. at zero time). Samples were removed at different times and specific activities of ribosomal protein fractions (core-30, core-50, split-30 and split-SO) both in soluble extract and in mature ribosomes were determined 8s desoribed in Materials and Methods. The Figure includes specific activities ver8u.g time plots for different ribosomal protein fractions; (8) core-30, (b) split-30, (c) core-50, (d) split-50. --O-O-, Soluble extract; -O-e--, mature ribosomes.

(6) Immunological reactionsof intact 30 s and 50 s particles with antisera directedagainst wre and split proteins Several reports (Traub & Nomura, 1969; Mizushima & Nomura, 1970) suggestthat core proteins are first addedto RNAin the assemblyof ribosomes.Craven & Gupta (1970) have tentatively classifiedproteins in the 30 s subunit as “internal” and “external”. Internal proteins in intact ribosomal subunits do not react with protein-modifying reagents and are presumed to enter early in the assembly process. We tested the immunological reactions of intact 30 s and 50 s subunits towards specific antisera directed against core and split proteins. In this experiment purified ribosomes were suspendedin standard low Mg ‘+ buffer. Ribosomesin this buffer completely dissociate into subunits. Antisera against core and split proteins derived from 30 s and 50 s ribosomal subunits were added separately to the above solution. The mixtures were incubated for 15 minutes at 3 to 4°C and then centrifuged at 10,000g for 10 minutes. The clear supernatant fractions were analysed by sedimentation in sucrosedensity gradients. As seenin Figure 7, the amounts of ultraviolet-absorbing material under the 30 s peak are considerably reduced in the presenceof antisera, directed against 30 s split and core proteins. Similarly, in the presenceof antisera against 50 s split and core proteins, only the peak corresponding to the 50 s subunit is reduced. While these 20

292

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S. GUPTA

AND

U.

N.

RINGH

1.c (a) 0.E 0.E 0.7 0.E 0.5 0.4 0,: 0.2 E si=

0.1

s 0 !gj I.0 ‘Z : iJ 0.9

(b)

0.8 0.7

06

0.5 0.4

0.3 02 0.1 0

I 20 Fraction

no.

Fm. 7. Immunological reactions of intact 30 s and 50 s ribosomal subunits with antisera, directed against core and split proteins. In this experiment dissociated ribosomal subunits in standard low Mg2+ buffer were mixed with 0.1 ml. of different antisera and allowed to stand for 15 min. The reaction mixtures were then centrifuged at 10,000 g for 10 min, and supernatant fractions were analysed by sedimentation on sucrose density gradients for the relative amounts of 30 s and 50 s ribosomal subunits. (8) --X--X--, Normal serum; -O-O--, antiserum against SP-50; -@-a--, antiserum against core50.(b) --X--X--,Normal serum, .-Ok-a-,antiserumagainst SP-30;-O-O-, antiserum against core-30.

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observations once again clearly demonstrate the specificity of our serological preparations, they fail to detect any difference in the immunological reactions of intact subunits towards antisera directed against their core and split proteins. Of course, the possibility that only a few of the protein molecules present in our core protein preparation may be &&ally blocked, and hence synonymous with the internal proteins of Craven & Gupta (1970), cannot be excluded from our results.

4. Discussion (a) Free ribosomulprotein pooh in solublebacterial extracts Several workers in the past have postulated the existence of free ribosomal protein pools in bacteria. Nomura & Watson (1959) and Kurland & Maaloe (1962) on the basis of their observations on the accumulation of sub-ribosomal particles (CM particles) in the presence of chloramphenicol estimated that as much as 20 to 25% of total ribosomal proteins in bacteria may be present in the free state. Schleif (1967), on the other hand, estimated the amount of total precursor poolsto be lessthan 5%. Although values reported by other workers (Young & Nakada, 1970; Santer et al., 1968; Nakada, Anderson & Magasanik, 1964; Dalgarno & Gras, 1968) fall within this wide range, they show considerable variations among themselves. All these estimates have been inferred from indirect observations and in most casesno clear distinction can be made between ribosomal proteins existing in the free state and those associatedwith subribosomal particles. Values derived from kinetic studies on the incorporation of labelled precursors are perhaps physiologically more meaningful as the system in this case is least altered. However, in view of its complex nature, such analyses of the kinetics are often based on a number of assumptions which could be justified as a first approximation only, under certain experimental conditions. This, indeed, may be responsible for wide variations in the values reported from different laboratories (Schleif, 1967; Sells & Davis, 1970; Cozzone et al., 1969; Gierer t Gierer, 1968). In the results presented in this paper, we have divided the structural proteins of ribosomesinto four classes.This division is purely operational in the sensethat it is based on the relative easewith which different proteins are releasedfrom the nucleoprotein complex by high salt concentration in the presence of urea. In vitro reconstitution of ribosomal subunits has indicated that whereas core fractions may consist predominantly of proteins which are attached to RNA during early stagesof maturation, split proteins are added at later stages(Mizushima & Nomura, 1970; Traub & Nomura, 1969). Recently, Homann & Nierhaur (1971) have compared proteins from core particles with those from in vivo sub-ribosomalparticles and observedconsiderable similarity between them. In our isolation procedure, about 50% of the total ribosomal subunit proteins were released as split proteins. Values presented in Table 2 refer specifically to free ribosoma1proteins in soluble extract. The latter has been subjected to extensive centrifugation to ensure against any contamination by subribosomal particles. Contamination by non-ribosomal proteins in our immune-precipitation is estimated to be lessthan 10%. It is concluded that as much as 7 to 9% of total ribosomal proteins may exist in the free state. Values reported by earlier workers (20 to 25%) are probably too high and may be attributed to side effects of chloramphenicol, which has been shown to cause extensive degradation of pre-existing ribosomesin E. coli (Young & Nakada, 1971; our unpublished observations).

294

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Proteins in 50 s and 30 s subunits are known to consist of about 34 and 20 distinct molecular species, respectively. It has been further suggested that a ribosome could accommodate only one molecule of each type. Traut d al. (1969) have carried out detailed analyses of molecular weights and relative amounts of various proteins present, in 50 s and 30 s subunits. The total molecular weights of proteins in 50 s and 30 s particles, assuming one copy of each type of protein is associated with these subunits, have been estimated by these workers to be 590,000 and 413,000 daltons, respectively. Thus the weight ratio of proteins in 50 s to those in 30 s is 1.43. Measurements on the amounts of proteins further suggest that relatively larger number of proteins in 30 s (7 in 30 s compared to 2 in 50 s) are present in amounts significantly less than that expected on the basis of one copy per subunit. It is not clear at present whether these proteins constitute a distinct class, having only transient association with ribosomes depending on their functional state, or they are preferentially leached out during isolation of the subunits. In either case, varied estimates of the ratio (50 s : 30 s proteins) reported in literature cited will tend to be higher than 1.43 due to the presence of relatively more labile proteins in 30 s particles. It is interesting to note that the average value (1.44 &O-06) of this ratio for the corresponding free proteins in soluble extract as estimated from the data in Table 3 is comparable to 1.43. These observations strongly suggest that ribosomal proteins are present in a cell in equimolar amounts and that they are synthesized in a co-ordinate manner. As mentioned earlier, mature 50 s and 30 s subunits contain equal amounts of split and core proteins. In contrast, the amount of free core proteins in soluble extract has been consistently found to be smaller than that of free split proteins. It is further observed that, in kinetic studies the time-course of incorporation of radioactive amino acids (Fig. 5), specific activities of core proteins tend to be higher than that of split proteins. These observations again support our contention that ribosomal proteins are synthesized in a co-ordinat’e manner. Relatively smaller pool size and higher specific activities of core proteins are consistent with such a mechanism and are probably due to the fact that most of the proteins comprising this fraction appear as sub-ribosomal particles in a cell (Homann & Nierhaur, 1971). (b) The two major

components

of ribosomes, proteins co-ordinate manner

and RNA,

are synthesized

in n

It is well known that the two major RNA components of ribosomes,23 s and 16 s RNA, are present in equimolar amounts in exponentially growing bacteria under different growth conditions. The observations presented in this paper on the size and kinetic properties of free ribosomal protein pools led us to postulate that the large number of distinct molecular speciescomprising these proteins may also be synthesized in a co-ordinate manner. It is not too difficult to visualize possiblemechanisms of co-ordinate synthesis of several molecular speciesbelonging to the same class of macromoleculeswithin the conceptual framework of our present understanding of the transcription and translation processes.For example, polycistronic messengersor isolated cistrons under the control of common regulatory elements may provide an adequate mechanism for the synthesis of several protein molecules in a co-ordinate manner. In recent years synthesis of ribosomal RNA has been extensively investigated. There are a number of possiblemechanismswe may consider. (i) 16 s and 23 s RNA’s constitute a single operon and are first transcribed as a polycistronic RNA. During

BIOGENESIS

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maturation the precursor molecule is then split into 16 s and 23 s RNA by an appropriate endonuclease.Ribosomal RNA in higher organisms appears to be synthesized by this mechanism. However, no heavy precursor molecule comparable to that obtained in animal cellshas been detected in bacteria. (ii) The two isolated cistrons may be considered to possessindependent but identical initiation sites for RNA polymer&se.(iii) We may consider cistrons corresponding to 16 s and 23 s RNA in adjacent positions but with only one initiation site for both of them. There is someexperimental evidence supporting (iii), where the common initiation site is located at a site in DNA corresponding to the 5’-end of 16 s RNA; the cistron corresponding to 23 s RN9 being situated in an adjacent position at the 3’-end of 16 s RNA. Thus the same polymerase first transcribes 16 s RNA and then 23 s RNA (Pato & Von Meyenberg 1970; our unpublished observations). In all the three mechanismsoutlined above, the only constraint imposed on the system is that transcriptions of both 16 s and 23 s cistrons are initiated with the same frequency. We wish to emphasizethat this contains both the necessary and su3icient condition for a co-ordinate synthesis of two ribosomal RNA’s. Recently, Mangiarotti et al. (1968) concluded that transcription times for both 16 s and 23 s are the samein bacteria. This conclusion is primarily based on the observed lag period (as measured by an extrapolation of the linear incorporation rate in the steady state, to the time axis). We believe, as has been pointed out by Adesnik & Levinthal(1969), that a lag in an isotopic incorporation experiment could also be due to delay in equilibration of intracellular pools with exogenous labelled precursors. Indeed, it can be readily demonstrated on the basis of the formulation developed by Mangiarotti et al. (1968) that a higher incorporation rate for 23 s RNA is not inconsistent with its longer transcription time. Assuming the frequency of transcription as well as rate of movement of polymerase to be the same for both 16 s and 23 s RNA cistrons, equations (4) and (5) of Mangiarotti et al. (1968) can be rewritten in slightly modified form: R* = int2

for

0

(4)

and R*=inL2+nL(t-LL)=nLt--inL2

for

t > L,

(5)

where n is distance between adjacent polymerase molecules expressed as number of nucleotides; L is transcription time. For simplicity, the unit of time is defined here as the time taken by polymerase to traverse the distance n. It is evident from equation (5) that, in the steady state, the incorporation rate will be directly proportional to the transcription time, or the length of RNA. The question as to how rates of synthesis of ribosomal RNA and proteins are coordinated in the cell still remains unresolved. The earlier notion of ribosomal RNA functioning as a polycistronio messengerin the synthesis of ribosomal proteins, appeared quite feasible, though it now seemsuntenable for a number of reasons. This aspect has been discussedin detail by several workers in the past (Sypherd, 1967; Mangiarotti & Schlessinger,1967; Manor $ Haselkorn, 1967) and it is not necessary to go into it here. Failure to detect free ribosomal RNA in bacteria has led to the conclusion that ribosomal proteins may get attached to the nascent RNA before the latter is releasedfrom the DNA template. It is conceivable that such attachment, far from being a passiveprocess,may have a positive role in the progressof transcription

396

R.

S. GUPTA

Growth

FIQ. 8. Relative amounts conditions. D8t8 presented in Table amounts of non-ribosomal bscteri81 growth (-X-X-). ribosomes (-•--•-).

of free and bound

AND

U.

N.

SINGH

rate (min-‘x10’)

ribosomel

proteins

in

E. coli under

different

growth

3 are plotted here 8s the rstio of 8mounts of free ribosomsl proteins/ proteins in soluble extrects versus the exponential rete constant for The Figure also includes a similar plot for proteins in mature

of ribosomal RNA. This viewpoint is compatible with the existence of a large amount of free ribosomal proteins in growing bacteria as observed in these studies. The role ascribed to the ribosomal proteins in facilitating the transcription of ribosomal RNA envisaged in this model is analogousto that of ribosomesin the synthesis of messenger RNA postulated by Stent (1967). Phenomenologically, the two mechanisms are identical in the sensethat they enable a cell to maintain a balance between various elements belonging to the samefunctional unit. While Stent’s model ensuresagainst production of messengerRNA in excessover the capacity of translational machinery available in a cell, the second mechanism maintains a balance between ribosomal RNA and proteins which constitute elements of a common functional unit, i.e. a ribosome. It is conceivable that one of the protein components may also positively regulate the amount of ribosomal RNA in a cell by controlling the initiation of its transcription. In fact, Travers, Kamen & Schleif (1970) have isolated a protein factor from ribosome-free crude extracts of E. coli which is essential for ribosomal RNA synthesis in vitro. The possibility that this factor is one of the ribosomal proteins deserves serious consideration. Maalee and his co-workers (Maalee & Kjeldgaard, 1966)in recent years have carried out extensive investigations into changesin the relative amounts of various macromolecular components in bacteria under different growth conditions. An important conclusion derived from these studies was that the ribosomal content expressed as the ratio of the amount of ribosomal RNA or protein to the tot4 massof bacteria is directly proportional to the exponential growth rate constant. In Figure 8 data from Table 3 are plotted as the ratio of the amount in free ribosomal protein pools to that of non-ribosomal proteins in soluble bacterial extracts, against exponential growth rate. The Figure also includes a similar plot, namely, mature ribosomal proteins/non-ribosomal proteins ver.su.sgrowth rate. It is interesting to note that the amounts of free as well as bound ribosomal proteins increase in a parallel manner. Theseobservations are consistent with the mechanismoutlined above,

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where we have ascribed a positive role to free ribosomal proteins in facilitating the transcription of ribosomal RNA. However, the question as to how the differential rate of synthesis of ribosomal proteins is regulated is still an open one. Mechanisms proposed (Maalee, 1969; Koch, 1970) to account for these variations have been based primarily on empirical considerations. Assuming that the basic processesinvolved in the synthesis of ribosomal proteins are essentially similar to those involved in the synthesis of non-ribosomal proteins, we have examined various theoretical implications of the role of free ribosomal protein pools as a positive control element in the biogenesisof ribosomes,on the basisof a model proposedearlier (Singh, 1969; Singh & Gupta, 1971). The analysis has provided a rational basis for the observed variations in free and bound ribosomal proteins with growth rate (to be presented in a separate communication).

Appendix A quantitative analysis of kinetic data on the incorporation of lubelledamino acids into ribosomes Kinetic curves shown in Figures 5 and 6 are qualitatively consistent with a precursor-product relationship between ribosomal proteins present in free and bound states. We have carried out a detailed quantitative analysis under certain simplifying assumptions. The analytical method used in these calculations has been successfully applied to an analysis of ribosomal RNA synthesis in higher organisms(Pandharipande & Singh, 1968). For the sake of simplicity, we assumethat free ribosomal protein pools defined in these studies are kinetically homogeneous,and that in exponentially multiplying bacteria all cellular components increase exponentially with the same rate constant as that of bacterial growth. Under these conditions, the differential equation for the rate of change of specific activity (8,) of the free ribosomal protein pool with time can be written as: dfJP --- Pal,82 (fg+ggpp, (14 dt P where 8, is specific activity of amino-acid pool, P is amount of free ribosomal proteins, papis amount of amino acids incorporated into protein per unit time and ppris amount transferred from protein precursor pool to mature ribosome per unit time. In the steady state, Par, -=I

P Similarly, variation in specific activity is given by:

“;: +gg. (8,) of proteins in mature ribosome with time

If R is the amount of proteins in mature ribosomes then, y = % = ig

i.e. the

exponential rate constant for bacterial growth. Note that rates (p,, and p,,) and amounts (P and R) defined above refer to a constant volume of bacterial culture.

298

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Equations (1A) and (2A) provide the rationale for the kinetic method used by earlier workers to estimate the half-life of ribosomal precursor pools, and enable us to define precisely the conditions under which such estimates can be obtained. Assuming that the exogenous labelled amino acids are rapidly equilibrated with intracellular amino-acid precursor pools, and that the specific activity of the latter remains constant during the period of observation, equation (1A) can be readily integrated and written as : S, = S, - (S, - S,(O)exp(--0lt).

(3A)

Thus, under these conditions the incorporation of radioactive amino acids into free ribosomal protein pools (8, vers~.~ time curves in Fig. 5) may follow first-order kinetics, and the rate constant can be readily evaluated by conventional methods for curve analysis. It is evident that if the conditions implicit in the derivation of equation (3A) are fully satisfied, then specific activities of free proteins in different fractions should approach a constant value, i.e. that of amino acids in the cell. As seen from Figure 5, although specific activities of proteins at saturation are of the same order of magnitude, they are not exactly equal. It is not obvious whether this discrepancy is due to the kinetic heterogeneity of free ribosomal protein pools or due to the complex nature of the precursor used. Also, the possibility that the constraints imposed on the hypothetical model are not strictly valid for the real system, cannot be excluded a priori. On the basis of the observed specific activity values at saturation, the average half-life of free ribosomal proteins is estimated to be about 50 seconds. This appears to be much shorter than that (6 min) reported by Sells & Davis (1970). The usefulness of pulse-and-chase studies in macromolecular synthesis depends primarily on rapid dilution of radioactivity in low molecular weight precursor pools, which is normally ensured by adding a large amount of unlabelled chaser to the medium. The dilution is brought about by rapid exchange of molecules across the cellular membrane together with an ‘expansion’ of the intracellular pool due to increased uptake. In the quantitative interpretation of data derived from such studies, it is tacitly assumed that dilution of radioactivity due to the replenishment of the pool by endogeneously synthesized precursors from alternative nutrients in the medium, is a relatively slow process and hence negligible. Consider an idealized system in which the specific activity of amino acids in the cell is reduced to a constant value S, (co) immediately after the addition of chaser. 8, is then given by: (4A) 8, = & (00) + V,(O) - 6 (00)) {exp(--~)L where S,(O) = specific activity of ribosomal protein at zero time, i.e. at the time of addition of cold amino acids as chaser. Integration of equation (5A) in this case will lead to an expression for 8, containing two exponential terms 01and y. It can be shown that only when CLB y < 1 can the expression for S, be approximated to first-order kinetics and is given by:

4 = 440) + ysp(o~ I:‘“’

(1 - exp(-at)}.

Thus under certain assumptions, which are implicit in the derivation of equation (5A), the half-life of ribosomal precursor pools can be determined from the observed incorporation of radioactive amino acids into mature ribosomes in a pulse-and-chase experiment. This, indeed, has been the rationale behind the studies reported by earlier workers (Schleif, 1967; Davis & Sells, 1969).

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In contrast to the wide variations in half-lives of different ribosomal proteins observed by Sells & Davis (1970) and Davis & Sells (1971), the values of LYobtained in the present studies appear to be of the same order of magnitude for all the four classesof proteins. The average half-life (- 80 set) derived from the observed incorporation of radioactivity into mature ribosomal proteins iu a pulse-and-chaseexperiment (equation (5A), Fig. 6) is slightly longer than that (II 50 set) estimated from data in Figure 5. Au important point which must be emphasized here is that the former estimate bears a remarkable resemblanceto the average half-life (N 76 set) inferred from the lossof radioactivity in a free ribosomal protein pool in the sameexperiment (Fig. 6). The latter value was obtained from the slope of the linear plot S,-S, (w) z’er
S, = S,(O) + y[fV - k 6% - S,(O)) (1 - exp(-411.

t

(64

3

Split 50

FIG. 9. Kinetic data on the time-course of incorporation of labelled amino acids into various classes of ribosomal proteins presented in Fig. 5 were analysed on the basis of a preoursor-product relationship between proteins in free and bound form. The theoretical basis for these analyses is desoribed in the Appendix. In these Figures, specific activities (8,) of various classes of proteins in mature ribosomes at different times are plotted against X, where x

=

s

t a

_

8%

-

SIJ(W

‘2

{1 -

(exp

-

at)}

Values of S,(O), S, and LYwere calculated from the observed incorporation of radioactive amino anids into free ribosomal protein pools &s shown in Fig. 5. Values of y were estimated from the slopes of the linear segments of these curves. The physiological significance of this parameter is disoussed in detsil in the text.

300

R. S. GUPTA

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This enables us to determine y, which can be readily obtained from the slopeof t,he linear plot, S, versus X, where X = Sat - s~-~sp(o){l

- exp(-at)}.

Data from

Figure 5 plotted as S, versu.sX are shown in Figure 9. The deviation from linearit’y in the later part of the curves is probably due to the approximation S, B S,, which is valid only during the early period of incorporation. The values of y estimated from the slopesof the linear segmentsof the curves in Figure 8 for the four classesof ribosomal proteins are found to be 0.05 mine1 (core-30), 0.12 min-l (core-50), 0.12 min-1 (SP-30) and 0.09 min-l (SP-50). As pointed out earlier, for an idealized model system, y should be equal to the exponential growth rate constant of the bacteria. The rate constant 0.012 min-l inferred from the observed doubling period of the bacteria (60 min) is much smaller than the values of y calculated from incorporation studies. The reasonsfor this discrepancy are not clear at present. While the discrepanciescould be due to the simplifying assumptions implicit in these calculations-and this cannot be excluded a priori-it is also likely that the bacterial cultures in our studies have not yet achieved a steady state of exponential growth. The size of the ribosomal precursor pool has often been estimated from its half-life and the observed bacterial growth rate. In the present studies, assumingthe bacterial growth rate to be 0.012 min-I, corresponding to a doubling period of 60 minutes, and the value of (Yto be 0.81 min-I, the ratio of ribosomal protein pools to mature ribosomeisfound to be 0*012/041= 0.015. This is much smallerthan the figure of 0.086 obtained from the immuno-precipitation data (Table 3). However, if we consider an average value of y to be 0.1 min- l, as estimated from kinetic data on the incorporation of labelled amino acids, the value of this ratio, 0.12, is of the same order of magnitude as that obtained from immuno-precipitation. This not only adds to our confidence in the analytical method used in these studies, but also suggeststhat the observed high values of y in comparison with the bacterial growth rate may have physiological implications.

REFERENCES Adesnik, M. t Levinthal, C. (1969). J. Mol. Biol. 46, 281. Bray, G. A. (1960). Analyt. Biochem. 1, 279. Cozzone, A., Marvaldi, J. & Marchis-Mouren, G. (1969). Biochemietry, 8, 4709. Craven, G. R. & Gupta, V. (1970). Proc. Nut. Ad. Sci., Wash. 67, 1329. Dalgarno, L. & Gros, F. (1968). Biochim. biophye. Acta, 157, 52. Davis, F. C. & Sells, B. H. (1969). J. Mol. Biol. 39, 503. Davis, F. C. & Sells, B. H. (1971). Biochim. biophys. Acta, 232, 379. Ecker, R. E. & Schaechter, M. (1963). B&him. biophye. Acta, 76, 275. Gierer, L. & Gierer, A. (1968). J. Mol. Biol. 34, 293. Godson, G. N. (1967). In Methods in Enzymology, ed. by L. Grosman & K. Moldave, vol. 12A, p. 503. New York: Academic Press. Guthrie, C., Naahimoto, H. & Nomura, M. (1969). Proc. Nut. Acud. Sci., Wash. 63, 384. Homann, H. E. & Nierhaur, K. H. (1971). Europ. J. Biochem. 20, 249. Hosokawa, K. & Nomura, M. (1965). J. Mol. BioE. 12, 225. Itoh, T., Otaka, E. & Osawa, S. (1968). J. Mol. BioZ. 33, 109. Kennel, D. & Magasanik, B. (1962). Biochim. biophys. Acta, 55, 139. Koch, A. L. (1970). J. Theor. BioZ. 26, 203. Kurland, C. G. & Ma&e, 0. (1962). J. Mol. BioZ. 4, 193. Lewandowski, L. J. & Brownstein, B. L. (1969). J. Mol. BioZ. 41, 277.

BIOGENESIS Lowry,

0. H., Rosebrough,

OF RIBOSOMES

N. J., Farr, R. J. & Randall,

IN E.

301

COLI

R. J. (1951). J. Bid.

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