Biosynthesis of 50 s ribosomal subunit in Escherichia coli

J. Mol. Biol. (1969) 40, 321-351

Biosynthesis of 50 s Ribosomal Subunit in Escherichia coli SYOZO OSAWA, EIKO OTAKA,

TAKUZI

Pront AND TAKESHI

FUKUI

Department of Biochemistry and Biophysics, Research Institute for Nuclear Medicine and Biology, Hiroshima University Kasumi-Cho, Hiroshima, Japan (Received 26 May 1968, and in revised form 30 September 1968) Two intermediate particles of 50 s ribosome formation, i.e. [30 s]-$ and 40 s were isolated from exponentially growing nascent or precursor particles, Escherichia coli cells. Particles indistinguishable from these nascent ribosomal particles were found in the extracts of the cells treated with a low concentration of chloramphenicol. The [30 s]-particles contain undermethylated 23 s rRNA and three main protein components. The 40 s particles also possess undermethylated 23 s rRNA and, in addition to the three protein components found in the [30 s]-particles, 9 other components out of 19 components detectable in the 50 s ribosomal subunit. Both particles include little 5 s RNA. Based on the above findings, a scheme for the biosynthesis of 50 s ribosomal subunits is presented. The protein compositions and 5 s RNA contents of the nascent ribosomal particles are compared with those of protein-deficient particles obtained by exposing 50 s ribosomal subunits to a concentrated LiCl solution. The molecular compositions of these two kinds of particles are found to be similar, but not exactly the same.

1. Introduction It has been proposed that the 50 s ribosomal subunit is formed via two sequential intermediates of 30 to 32 s ([30 s]-nascent ribosomal particles$) and 40 to 43 s (40 s nascent ribosomal particles) (Roberts, Britten & McCarthy, 1963; Mangiarotti, Apirion, Schlessinger & Silengo, 1968; Osawa, Otaka, Muto, Yoshida & Itoh, 1967). These nascent ribosomal particles may be demonstrated by sucrose-gradient centrifugation of crude extracts prepared from growing Escherichia coli cells pulse-labelled with

isotopic

precursors.

A considerable

amount

of similar

particles

has been detected

in the extracts from the cells treated with low concentrations of chloramphenicol (LCM-particles of Osawa et al., 1967; Otaka, Itoh & Osawa, 1967). The low CM& particles are considered to be identical with the nascent ribosomal particles, since they are indistinguishable from each other in their sedimentation coefficients, nature of the component rRNA included, protein compositions and 5 s RNA contents, as will be reported in this paper. The protein and 5 s RNA analyses of the [30 s]- and 40 s nascent ribosomal particles have provided information concerning the sequence t Present address: Laboratory of Plant Pathology, Faculty of Agriculture, Nagoya University, Chikusa-Ku, Nagoya, Japan. $ The 30 s particles containing 23 s rRNA will be referred to as [30 s]-particles in order to distinguish them from 30 s ribosomal subunits having 16 s rRNA. $ Abbreviation used: CM, cbloramphenicol. 321 28

322

8. OSAWA,

E. OTAKA,

T. ITOH

AND

T. FUKUI

with which a series of ribosomal proteins is assembled and the step where 5 s RNA is inserted during the formation of the 50 s ribosomal subunit. It has been demonstrated also that the protein compositions of the nascent ribosomal particles are closely similar to, but not exactly identical with, those of the protein-deficient particles produced by exposing 50 s ribosomal subunits to a high concentration of LiCl (Li-particles of Itoh, Otaka & Osawa, 1968). Preliminary notes of some of the above results have been published (Osawa et al., 1967; Otaka et al., 1967).

2. Materials and Methods (a) Organ&n

and culture conditions

Cells of Eecherichia co& B(H) were used throughout. The bacteria were inoculated in a minimal Tris-salts-glucose medium (usually 50 ml.) (Watanabe, 1957) at a cell density of about 1.3 x lOs/ml., and grown under aeration at 37°C. (b) LabeUing of protein and RNA

with radioactive isotopes

Three types of labelling were performed for the preparation of labelled subribosomal particles and ribosomal subunits. (i) Growing cell cultures at a cell density of 4.5 x lOs/ml. received [3H]lysine (10 &ml. culture plus 5 pg of non-labelled lysine/ml.), [3H]uridine (2 &ml. culture plus 20 pg of non-labelled uridine/ml.) or [3H]uridine plus L-[methyZ-14C] methionine (0.6 @/ml. plus 2 pg of L-methionine/ml.) together with 0 to 1.5 pg of CM/ml. (usually 0.8 rg/ml.), The incubation was carried out for 20 min (low CM-cells). (ii) The bacteria were grown until the cell density reached 4.5 x lOs/ml. The culture then received a mixture of L-[3H]lysine, L-[“Hlvaline and L-[3H]leucine (each 20 PC/ml. culture), [3H]uridine (2 &ml. culture), or [3H]uridine plus L-[maethyZ-14C]methionine (1 PC/ml.) for 60 to 300 sec. The cell suspension was poured on to crushed ice to terminate the incubation (pulselabelled cells). (iii) About 30 min after the inoculation of bacteria, the culture received 32P (50 &ml. culture) or [3H]uridine (2 &ml. culture plus 20 IL@;non-labelled uridine/ml.) plus L-[methyZ-14C]methionine (0.6 &ml. plus 2 pg L-methionine/ml.) to label RNA, or a mixture of [3H]lysine, [3H]arginine, [3H]valine and [3H]leucine (each 10 PC/ml. culture plus 15 pg each non-labelled amino acid/ml.) to label protein. The incubation was continued for 20 min to 3 generations according to the purpose. All the cell suspensions were collected by centrifugation, washed with 10-z M-Tris-HCl containing lo-* M-magnesium acetate at pH 7.8 (Tris-Mg buffer) and frozen at -20°C until use.

(c) Isolation

of various

aubribosomul

particles

and ribosomal

subunita

Frozen cells were ground with quartz sand and mixed with a small amount of Tris-Mg buffer solution containing 5 fig DNase/ml. When the protein composition was analysed for the nascent ribosomal particles from exponentially growing cells, it was found necessary to grind the cells with about 3 mg ( = 0.3 ml.) of nascent rRNA to absorb non-ribosomal proteins (see Results). The mixture was centrifuged at 9000 rev./min. The supernatant was used as crude extract. The extracts were then applied to a sucrose-Tris-Mg buffer solution (5 to 20% linear sucrose gradient) and centrifuged in an SW25.1 or SW25.3 rotor of a Spinco L2 preparative ultracentrifuge at 4°C at 21,000 rev./min (SW25.1) or 22,000 rev./mm (SW25.3) for 17 hr. After centrifugation, the tube content was fractionated at 4°C into 40 F 1 fractions. A 0*03-ml. portion of each fraction was placed on a filter disc, dried and the radioactivity was determined to obtain sedimentation profiles (Figs l(a) and 2(a)). The radioactive material in the region between 50 and 30 s (40 s region) was separately dialysed against Tris-Mg buffer for 5 hr at 4°C to remove sucrose, and was subjected to a second sucrose-gradient centrifugation. The 40 s region gave three components, i.e. 40 s particles, 60 and 30 s ribosomal subunits (Figs l(b) and 2(b)). The 60 s ribosomal subunits and 40 s particles so prepared from the three types of cells described in (b) were used for further analyses. The approximately 30 s material obtained after the iirst sucrose gradient centrifugation was further fractionated on a Sepharose 4B column

FIG. 1. Purification

(a)

I

30

1

20 0

of [r4C]lysine-labelled

Tube no. (b)

IO

,

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30

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(c)

40

[30]- and 40 s low CM-particles.

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Exponentially growing cells in 50 ml. culture medium received 500 PC of [3H]lysine plus 260 pg of non-labelled lysine and 0.8 pg of CM/ml. The incubation was continued for 20 min at 37°C. After harvesting the cells, an extract was prepared with Tris-Mg buffer containing 6 pg D&se/ml., and centrifuged on a sucrose gradient in an SW25.1 rotor at 21,000 rev./min for 17 hr at 4°C. After fractionation, a 0.03-ml. portion from each fraction was placed on a filter disc and the radioactivity was measured (a). The materials in the 40 s region indicated by ( 0, in (a)) were dialysed against Tris-Mg buffer to remove sucrose and again centrifuged on a sucrose gradient at 20,000 rev./min for 19 hr at 4°C (b). The tube contents (0, in (b)) were pooled and served for the protein composition analyses of the 40 s low CM-particles. The materials in the 30 s region shown by (0) in (a) were concentrated to about 1 mI. and then fractionated with a coIumn of Sepharose 4B (c). The fractions in the region of [30 s]-low CM-particles shown by ( 0) in (c) were then centrifuged on a sucrose gradient at 23,000 rev./min for 18 hr at 4°C (d). The material in the regions shown by (0) in (d) was used for the protein composition analyses of the [30 s]-low CM-particles.

IO

[3Osl

324

S. OSAWA,

E. OTAKA,

(a)

T. ITOH

AND

Tube no.

T. FUKUI

(b)

FIG. 2. Purification of radioactive 40 s nascent ribosomal particles from exponentially growing oells homogeneously labelled with a mixture of E3H]amino acids. Exponentially growing cells (50 ml.) were incubated with a mixture of lysine, arginine, valine and leucine all labelled with 3H (each 600 PC) plus 750 pg of each non-labelled amino acid for about 3 generations. After harvesting the cells, 3 mg (=0.3 ml.) of nascent rRNA were added and the cell extract was prepared as described in the legend to Fig. 1. It was centrifuged in a sucrose gradient at 21,000 rev./min for 17 hr at 4% in an SW251 rotor (a). The material in the region of 40 s indicated by (a) in (a) was dialysed and subjected to the second sucrose-gradient centrifugation at 21,000 rev./n& for 19 hr (b). The fractions shown by (0) in (b) were used for the protein composition analyses of the 40 s nascent ribosomal particles.

(Fig. l(c)) (Sepharose was obtained from Pharmacia Co., Uppsala, Sweden). About 1 ml. of the sample was applied on top of the column (1.0 cm x 100 cm) and then &ted with Tris-Mg buffer at a flow rate of 1 ml./20 min/tube at 4°C. The first peak on the chromatogram, which includes mainly [30 s]-particles, was again centrifuged in a sucrose gradient at 23,000 rev./min for 18 hr to obtain the purified [30 s]-particles (Fig. l(d)). The [30 s]particles could be prepared from low CM- or pulse-labelled cells. When the cells were labelled for periods exceeding 20 mm, the amount of labelled [30 s]-particles was very small as compared with the labelled 30 s ribosomal subunit end could not be satisactorily used for further analyses. The 40 s LiCl-particles were prepared by exposing 50 s ribosomal subunits (labelled with [3H]lysine or [14C]adenine) to 1.6 M-Lick@1 M-magnesium acetate according to the procedure previously described (Itoh et al., 1968).

(d) Preparation

ati

fractionation

of riboscwd

protein

The procedure described previously (Otaka, Itoh & Osawa, 1968) was used with some modifications. Purified particles labelled with [3H]- or [14C]amino acids were dialysed overnight at 4°C against 2 x 10e4 M-magnesium acetate (pH 6.8) to remove Tris and sucrose and freeze-dried. The dry particles were dissolved in 20 ml. of 5x 10m3 M-acetate buffer (pH 6.6) containing 2.5 x low3 M-EDTA and 6 M-urea and mixed with [3H]- or [14C]lysine-labelled 60 s ribosomal subunits as reference. The mixture was digested with 6 pg/ml. each of pancreatic RNaee and T, RNase at 37°C for 20 min in the presence of 3 mg of ribosomal protein as carrier. The sample was then adsorbed on to a 0.5 cm x 40 cm column of CM62 (Whatman’s carboxymethyl cellulose CM62, microgranular-preswollen, H. Reeve Angel & Co.) which had been equilibrated with 6 m-urea-5x 10m3 M-acetate buffer (pH 6.6). Elution was csrried out as previously described (Otaka et al., 1968).

BIOSYNTHESIS (e) Preparation

OF 60 a RIBOSOMES and fractionation

325

of RNA

The purified particles or ribosomal subunits were diluted threefold with Tris-Mg buffer solution containing O*5o/osodium lauryl sulphate and about 4 mg of E. coli total RNA as carrier. The mixture was shaken with an equal volume of 90% phenol at 4°C for 15 min. After centrifugation, 3 vol. of cold ethanol and a few drops of 4 M-NaCl were added to precipitate RNA. The ethanol suspension of the RNA was stored at -20°C until use. To determine the methyl group contents in 23 s rRNA, the RNA samples doubly labelled with [3H]uridine and [~thyZ-14C]methionine were dissolved in 1.5 ml. of Tris-Mg buffer, layered on top of sucrose-Tris-Mg buffer solution (2.5 to 15% sucrose gradient) and centrifuged at 4°C for 18 hr in an SW25.1 rotor. To separate 5 s RNA from 23 s (or 16 a) rRNA, the procedure developed by Galibert, Larsen, Lelong & Boiron (1965), and successfully used by Galibert, Lelong, Larsen & Boiron (1967), Morel& Smith, Dubanau & Marmur (1967) and Knight & Darnell (1967) was used. The RNA collected by centrifugation from a sample of the above suspension wss dissolved in 0.5 ml. of 0.05 M-Tris acetate (pH 7.2) containing O*2o/o sodium lauryl sulphate. The RNA solution was then applied on top of a Sephadex GlOO column (1 cm X 100 cm) which had been equilibrated with the above Tris-acetate-sodium lauryl sulphate solution. The elution was done with the buffer-sodium lauryl sulphateat room temperature. About a l-ml. fraction was collected at a flow rate of 15 min/ml. To obtain 14C-labelled 5 s RNA which was used as reference in the analyses illustrated in Figure 14, a preparation of RNA from [14C]adenine-labelled 50 s ribosomal subunit was fractionated with a column of Sephadex GlOO as described above. The 14C-labelled RNA fractions eluted just after the main RNA peak (23 s rRNA) were precipitated with 3 vol. of ethanol. The RNA so obtained was believed to be 5 s RNA, since (1) its mobility was slower than that of 4 8 RNA when examined with disc electrophoresis (see Knight & Darnell, 1967), (2) it sedimented faster (approx. 5 s ) than 4 s RNA, and (3) its chromatographic position on the Sephadex GlOO column agreed well with that described in the literature (Galibert et al., 19651967; Morel1 et al., 1967; Knight & Darnell, 1967). (f) Radioactivity measurements Radioactivity in RNA and protein was measured in a model 2002 Packard liquidscintillation spectrometer by the procedures described by Muto, Otaka & Osawa (1966) and by Otaka et al. (1968), respectively. (g) Isotopes The following isotopes were used. L-[4,5-3H]lysine (108 me/m-mole), L-[3H]arginine (380 me/m-mole), L-[4,5-3H]leuoine (232 me/m-mole), n,L-[3H]valme (480 me/m-mole), nniformly labelled [3H]uridine (1230 me/m-mole), r.,-[14C]lysine-HC1 (7.6 ma/m-mole), n-[naethyZ-14C]methionine (50 me/m-mole), [2-14C]uracil (40.6 me/m-mole), [S-r4C]adenine (35 me/m-mole), and [3aP]orthophosphate, all obtained from the Radiochemical Centre, Amersham, England.

3. Results (a) Xedimentation coejkients and RNA components of nascent ribosomul particles related to the formation of 50 S riboscmul subunit The [30 s]- and 40 a nascent ribosomal particles could be most clearly labelled by exposing the exponentially growing cells to [3H]uridine for 60 to 90 seconds under our experimental conditions. (The 26 s and other slower moving components were also labelled. These are mainly precursors to the 30 s ribosomal subunit, un6nished rRNA or messenger RNA, and will be dealt with in a forthcoming paper.) With longer periods of Iabelling, the radioactive 30 s ribosomal subunits accumulated and the 30 s region became a mixture of three components, i.e. [30 s]-particles, 30 s ribosomal subunits and 26 s particles. These three kinds of particles could not be differentiated

326

S. OSAWA,

E. OTAKA,

T.

ITOH

AND

T. FUKUl

by sucrose-gradient centrifugation, but were partially separated from one anothrl by a column of Sepharose 4B (see Materials and Methods and the next section). To characterize the [30 s]- and 40 s nascent ribosomal particles, an extract of cells pulse-labelled with [3H]uridine for 60 seconds was prepared and fractionated by sucrose-gradient centrifugation (Fig. 3(a)). The regions I and II indicated in Figure 3(a) were separately dialysed against Tris-Mg buffer, mixed with a small amount of [14C]uracil-labelled 30 s ribosomal subunit, and centrifuged in sucrose gradients. As can be seen in Figure 3(b) and (c), each fraction gave a single main ribonucleoprotein peak together with some slower-moving components. The sedimentation coefficients of these two main particles were estimated to be 40 s and 30 s, respectively, as calculated from the position of reference 14C-labelled 30 s ribosomal subunit. The RNA was then prepared from the respective purified particles and was examined by sucrose-gradient centrifugation. Both particles contained solely 23 s rRNA (Fig. 3(d) and (e)) confirming the results reported previously (Iwabuchi, Kono, Oumi & Osawa, 1965; Mangiarotti et al., 1968; Osawa et aZ., 1967). As can be seen in Figure 3(a) and in the literature (Roberts et al., 1963; Mangiarotti et aZ., 1968; Iwabuchi et al., 1965), the [30 s]- and 40 s particles sedimented as 32 to 33 s and 43 s, respectively, in the first sucrose-gradient centrifugation of the labelled crude extracts. As shown in Figure 3(c) and (d), the sedimentation coefficients of the particles decreased to 30 to 28 s and 40 s upon their recentrifugation on sucrose gradients with a release of slower moving material. (b) Ribonucleoprotein particles accumulated in the presence of low concentrations of chloramphenicol (i) Effect of various concentrations of chloramphenicol on ribosome formation [3H]Uridine was introduced to exponentially growing bacterial cultures in the presence of various concentrations of CM (0 to 1 mglml.). After 20 minutes, the bacteria were harvested and extracted. The crude extracts were then fractionated by sucrose-gradient centrifugation. It was found that CM at concentrations above 3 pg/ml. inhibited ribosome formation completely and a considerable amount of the typical 18 s and 25 s CM-particles was found in such cell extracts (Yoshida & Osawa, 1968). At concentrations of CM between 0.6 and 1.5 pglml., various ribonucleoprotein particles of sedimentation coeflicients higher than typical CM-particles were detected in the cell extracts together with some mature 50 s and 30 s ribosomal subunits (Fig. 4(a)). These immature particles will be called low CM-particles in this paper. CM FIQ. 3. Sedimentation analyses of nascent ribosomal particles prepared from cells pulse-labelled with [3H]uridine for 60 sec. Exponentially growing cells (50 ml.) received [3H]uridine (100 PC) for 60 set at 37%. The extract was prepared with Tris-Mg buffer containing 5 pg DNase/ml. It was centrifuged at 25,000 rev./min for 17 hr at 4% in an SW25.1 rotor. After centrifugation and fractionation, a 0*03-ml. portion from each tube was placed on a filter disc and the radioactivity was measured (a). The radioactive regions from I and II indicated in (a) were both dialysed against Tris-Mg buffer for 4 hr at 2°C to remove sucrose, and again centrifuged at 25,000 rev./min for 17 hr at 4°C in an SW25.3 rotor with a small amount of 30 s ribosomal subunit labelled with [‘*C]uracil as a reference. After centrifugation and fra&ionation, a O-05-ml. portion from each tube was used for radioactivity measurements. (b) region I (40 5); (c) region II ([30 81). The 3H-labelled RNA prepared from 40 s and [30 s]-particles was mixed with a small amount of 23 s rRNA as reference, and centrifuged in suorose gradients at 23,000 rev./min for 20 hr at 4’C in an SW25.3 rotor. (d) RNA from 40 s particles; (e) RNA from [30 s]-particles. -.-.-, “H; --O--O--, l&C.

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328

8. OSAWA,

E. OTAKA,

T. ITOH

AND

T. FUKUI

Tube no. (a)

(b)

FIG. 4. Sedimentation analyses of low CM-particles Exponentially growing cells (50 ml.) received r3H]uridine (100 pc) for 20 min in the presence of 0.8 pg of CM/ml. Centrifugation, 21,000 rev./min for 15 hr at 0 to PC in an SW25.1 rotor (a). A small amount of crude extract labelled for two generations with [‘%]uracil was mixed with all samples as internal standards for 50 and 30 s ribosomal subunits. Their positions are shown by arrows in each Figure. Materials of the 40 s region in (a) was again centrifuged at 22,000 rev./min for 18 hr at 4% in an SW26.3 rotor in a sucrose gradient with a small amount of 50 and 30 s ribosomal subunits labelled with [14C]uracil aa a reference.

at concentrations below 0.5 ,ug/ml. allowed the formation of mature ribosomes, although small amounts of immature particles were still detected with 0.5 pg CM/ml. (ii) Sedimentation coegicients and RNA component of low CM-particles To examine in more detail each class of radioactive low CM-particles formed in the presence of 0.8 pg CM/ml. (and labelled with [3H]uridine), each fraction obtained by the first sucrose-gradient centrifugation of a crude extract was further fractionated, or the RNA component of each particle was analysed. The 50 s peak was indistinguishable from the 50 s ribosomal subunit labelled with [f4C]uracil added as a reference in sedimentation analysis in a sucrose gradient. Both particles contained only 23 s rRNA. The 40 s region and 22 s region sedimented as a single peak, the 40 s peak having 23 s rRNA (40 s low CM-particle) and the 22 s peak having 17 s rRNA, respectively (Fig. 4(a) and (b)). The 30 s region contained both 23 s and 16 s rRNA and gave a broad peak by sucrose-gradient centrifugation, suggesting this fraction to be heterogeneous. We have thus used a column of Sepharose 4Bt to fractionate the approximately 30 s materials with a reasonable separation into three components t The molecular compositions of nascent ribosomal particles and ribosomal subunits are probably not affeoted by the Sepharose 4B chromatography, since it was confirmed that the protein compositions and 5 s RNA contents of 50 s and 30 s ribosomal subunits were the same before and after the chromatography.

BIOSYNTHESIS

OF 50 s RIBOSOMES

329

(Fig. 5(a)). The first peak mainly consisted of the particles of 28 to 30 s (hereafter called [30 s]-low CM-particles) containing only 23 s rRNA (Fig. 5(b) and (0)). The materials in the second peak sedimented as 26 s and contained only 17 s rRNA (Fig. 5(c) and (f)). The third one consisted of 30 s subunit (and some contaminated 26 s particles) formed in the presence of a low concentration of CM, since its chromatographic position with Sepharose 4B columns coincided with that of 30 s subunit; it sedimented as 30 s and contained only 16 s rRNA (Fig. 5(d) and (g)). Thus, as in the case of nascent ribosomal particles, we could recognize two kinds of low CM-particles containing 23 s rRNA, i.e. [30 s]-low CM-particles and 40 s low CM-particles.

(iii) On the date of rRNA

of low chloramphenicol

particles

in the cells

We have shown that the protein part of 18 s and 25 s CM-particles is merely what has been combined “artificially” with nascent rRNA during the isolation procedure (Yoshida & Osawa, 1968). It is possible then that low CM-particles described in this paper are also the same kind of artifact. Experiments were designed to check this possibility. Three kinds of cells were prepared according to the scheme shown in Figure 6(a) and (b). Cells-I and cells-II received [3H]uridine for 15 minutes in the presence of 0.8 pg CM/ml. (low CM). The cells-II were harvested, extracted and examined by sucrosegradient centrifugation. The rRNA formed under these conditions is found in low CM-particles (plus some mature ribosomal subunit) (Fig. 6(b)). The cells-I then received an excess of non-labelled uridine to terminate the labelling of rRNA to be formed in the presence of low concentrations of CM. At the same time, 100 pg CM/ml. (high CM) and [14C]adenine were added to stop protein synthesis and to label rRNA to be synthesized after the addition of the high concentration of CM. The cells were incubated for five minutes and harvested. In the presence of high CM, protein synthesis in the cells is almost completely inhibited, and rRNA formed (and labelled with 14C) under these conditions is found as 25 s and 18 s CM-particles in the cell extract, as shown by the profile of the extract of cells-III in Figure 6(b). In Figure 6(a) and (c) are shown the sedimentation profiles of the cells-I. Profiles of the extracts from cells-11 and III are shown in Figure 6(b), where the two profiles are superimposed after separate sucrose-gradient centrifugations. There is no essential difference between Figure 6(a) and (b), except that in Figure 6(a) 14C-labelled 25 s CM-particles predominate in amount over 14C-!abelled 18 s CM-particles, while in Figure 6(b) the situation is reversed. Sucrose-gradient eentrifugations of phenol-extracted RNA from cells-I and cells-III revealed that the amount of 14C-labelled 25 s rRNA was larger than l*Clsbelled 16 s rRNA in the former cells while in the latter 14C-labelled 16 s predominated. The difference may be ascribed to the difference of t,he labelling conditions, i.e. cells-I were pretreated with low CM while cells-III were not. Thus there is no indication of the mixing of 3H-labelled rRNA and l*C-labelled rRNA, and they exist as low CMpart#icles, and 25 s and 18 s CM-particles in the extract of cells-I, respectively. If low CM-particles are the same kind of artifacts as CM-particles, as a result of combination between rRNA and protein during the preparation of cell extracts, both ‘V-high CM rRNA and 3H-low CM rRNA should oqually pick up the proteins, prozidcd that both rRNA’s are present in bhe same state in the cells. As a result, the sediment’ation profiles of 3H and l*C may become identical with each other except for 3H-counts in mature rRNA. The experiment described above is thus consistent

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BIOSYNTHESIS

OF

50 s HIBOSOMES

33 I

with the idea that low CM-particles are not the same kind of artifact as 25 s and 18 s CM-particles, and at least shows that the state of low CM-rRNA in the cells is different from that of high CM-rRNA. (iv) Conversion of low chloramphenicol-particles

to ribosomd

subunits

upon removal of

chloramphenicol

To know the fate of low CM-particles in the cells after removal of CM from culture media, the following experiments were done. An exponentially growing bacterial culture was incubated with [3H]uridine and O-8 pg CM/ml. for 20 minutes. The cells were collected on a Millipore filter and washed with Tris-Mg buffer to remove [3H]uridine and CM. They were then incubated in 20 ml. of Tris buffer, containing O-2 mMEDTA, for three minutes (Leive, 1965), and divided into three equal parts (each 5 ml.). Cells from the first part were collected for the zero time control and extracted. The second and third parts were poured into 50 ml. of a fresh Tris-salts-glucose medium containing [14C]adenine and an excess of non-labelled uridine with and without actinomycin D, respectively. Both cells were cultured for a further 15 minutes, harvested and extracted. As seen in Figure 7, RNA of low CM-particles was almost quantitatively converted to 50 s and 30 s ribosomal subunits upon removal of CM, even when further RNA synthesis was arrested by actimomycin D. These results indicate that the rRNA moiety of low CM-particles can be converted to mature ribosomes. In another experiment, exponentially growing cells were incubated with [14C]lysine and 0.8 pg CM/ml. for 15 minutes. The cells were collected on a Millipore filter, washed with Tris-salts medium to remove [14C]lysine and CM, suspended in buffer and divided into two equal parts. Cells from the first part (cells-A) were collected and extracted. The second part (cells-B) was poured into 50 ml. of a fresh Tris-saltsglucose medium containing an excess of non-labelled lysine. The cells were cultured for a further 15 minutes, harvested and extracted. As seen in Figure 8, the 14C-counts in the regions of low CM- and mature ribosomal particles (particle materials) present in the extract of the cells treated with the low concentration of CM were quantitatively recovered in 50 s and 30 s ribosomal subunits after the removal of CM from the culture medium. It was confirmed that 40% of the hot trichloroacetic acid-precipitable 14C counts in the particle materials of the extract of low CM-cells was due to immature particles (low CM-particles) since it was sensitive to RNase (1 pg/ml. at 15°C for 15 min). Mature 50 s and 30 s subunits were resistant under the same conditions. The same results were obtained when actinomycin D was added to the culture after the removal of the low concentration of CM. It may be noted that neither increase nor decrease in 14C counts was observed in the soluble fraction before and FIG. 5. Fractionation of the 30 s region with a column of Sepharose 4B. The 30 s region (labelled with [3H]uridine) obtained by the first sucrose-gradient centrifugation (Fig. 4(a)) was mixed with a small amount of 30 s subunit labelled with [‘*C]uracil and layered on top of a column of Sepharose 4B (1 cm x 100 cm). The elution was done at 4% at a flow rate of 1 ml./20 min/tube (a). A sample of the regions I, II and III in (a) was centrifuged in sucrose gradients at 22,000 rev./ min at 4°C in an SW253 rotor. (b) Region I; (c) region II; (d) region III. RNA was prepared from the regions I, II and III of (a) and centrifuged with 14C-labelled 16 s rRNA &9 a reference in sucrose gradients at 25,000 rev./min at 4’ C for 20 hr in an SW25.3 rotor. (e) RNA of region I; (f) RNA of region II; (g) RNA of region III. -.-a--, 3H; --o--o--, 14C.

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g VI

c 0 *

uridine/ml.) for 15 min. The A growing E. coli culture (200 ml.) was incubated with CM (0.8 pg/ml.) and [3H] uridine (130 PC plus 40 pg non-labelled oell suspension w&9 then immediately Gltered on a Millipore titer, washed successively with Tris-Mg buffer and 0.1 ra-Tris buffer (pH 8.0). To the cell suspension were added 20 ml. of lOma M-Tris buffer containing 0.2 mhr-EDTA at 37%. It was incubated for 3 min at 37%. A 5-n& portion was taken out and served aa a zero time control (a). Two portions of 6 ml. each were poured into 50 ml. of a fresh Tris-salts-glucose medium containing 1 pc of [14C]sdenine and 6 mg of non&belled &dine/ml., with (b) or without (c) 5 pg actinomycin D/ml. Incubation was carried out for 16 min. Crude extracts were quantitatively prepared from the above three kinds of cells with Tris-Mg buffer containing 5 pg DNase/ml. and centrifuged in !xxxoBe gradients at 22,000 rev./min for 18 hr at 4°C in an SW25.3 rotor. -O--O----, 3H; -.--a--, lrC.

0

b

5 1

IC)

1

S. OSAWA,

334

2.73~10~

T. ITOH

AND

7’. FUKUI

I

/

:

E. OTAKA,

ctdmin

I\

/

>

I.61 x IO5 cts/min

’

4i

2.54 x IO5 ctdmin

\/ “l~58x10s cts/min

’

Tube no. (a)

(b)

Fra. 8. Conversion of low CM-particles to 50 s and 30 s ribosomal subunits upon removal of chloramphenicol (II). An exponentially growing aulture (100 ml.) was incubated with [r4C]lysine (10 PC) and 0.8 pg CM/ml. for 15 min. collected on a Millipore filter, washed with Tris-salts medium and suspended in 12 ml. of the same medium. A 5-ml. portion was harvested (cells-A). Another &ml. portion was poured into 60 ml. of fresh Tris-salts-glucose medium containing 500 pg of non-labelled lysine/ml. and incubated for 16 min (cells-B). Crude extracts were quantitatively prepared from the above two kinds of cells with Tris-Mg buffer containing 5 pg DNasejml. Centrifugation in sucrose gradients at 21,000 rev./min for 1’7 hr in an SW25.1 rotor. (a) Cells-A; (b) Cells-B.

after the removal of CM. This suggests the lack of appreciable ribosomal protein pool in the soluble fraction. Next, the cells-A and cells-B were labelled with [3H]lysine and [14C]lysine, respectively. These two types of cells were extracted and subjected to sucrose-gradient centrifugation to separate low CM- and/or ribosomal particles (particle materials) from soluble proteins. The protein of the 3H-labelled and 14C-labelled particle materials was simultaneously analysed on a CM52 column (Fig. 9(a)).? Similarly, 3H-labelled soluble protein and 14C-labelled soluble protein were also analysed (Fig. 9(b)). The 3H-labelled protein profiles in the particle materials in cells-A were essentially similar to the 14C-labelled proteins in 50 s and 30 s ribosomes in cells-B. Moreover, the soluble proteins of cells-A showed a similar profile to that from cells-B. A protein component (X in Fig. 9(b)) and a small amount of material in the ribosomal protein region would not be true ribosomal proteins, since (i) these proteins neither decreased nor disappeared when ribosome synthesis was resumed by removal of CM and (ii) their chromatographic positions (except for X) did not exactly coincide with those of t In one experiment, cells-B were treated with actinomycin D to prevent further rRNA synthesis. The protein pro& of the particle materials was again indistinguishable between cells-A and cells-B.

n

Fraction

no.

_.. -- .-.. ..-...-.

.

..----.------ ---.----.------

PIG. 9. Chrom8tographic analyses on CM62 columns of protein componenta of the particle materials and the soluble fraction. An exponentially growing culture (50 ml.) was incubated with [3H]lysine (680 PC = 130 pmoles) and CM (0.6 pg/mh) for 15 min (cells-A). Another 504. oulture was incubated with [14C]lysine (10 ~‘0 = 130 pmoles) and CM (0.8 pg/ml.) f or 15 min. The 14C-18behed cells were collected on 8 Millipore filter, washed and further incubated with nor&belled lysine (500 pg/ml.) for 20 min (cells-B). Cells-A and B were extracted and the crude extracts were centrifuged in 8ucrose gr8cLients at 21,000 rev./min for 17 hr in an SW25.1 rotor to separate the particle materials and soluble proteins. Total amounts of 3H-18belkd p8rticle protein (or 3H-labelled soluble protein) and 14C-labelled particle protein (or 14C-18belled soluble protein) were mixed and fractionated on a CM62 oolumn. (a) Protein of the particle materials; (b) protein of the soluble fraction. -O--O--, ‘4C;-._.-,3H.

I

336

S. OSAWA,

E. OTAKA,

T. ITOH

AND

T. FUKUI

ribosomal proteins (this was demonstrated by co-chromatography of 14C-labelled soluble proteins of cells-A and 3H-labelled particle proteins of cells-B). These two quantitative and qualitative experiments suggest that the proteins which had been present in the region of low CM-particles were quantitatively converted to mature 50 s and 30 s ribosomal subunits and that there is no appreciable contribution of the soluble protein for the formation of 50 s and 30 s ribosomal subunits. (c) Protein compositions of various subribosonml particles and 50 S ribosomd subunit (i) Fractionation of 50 S ribosomal protein with CM52 columns In previous papers (Otaka et al., 1968; Itoh et al., 1968), we recognized 15 main and several minor or ill-defined protein components in 50 s ribosomal subunit by carboxymethyl cellulose column chromatography in conjunction with disc electrophoresis. The use of CM52 columns (see Materials and Methods) made it possible to resolve four more components as definite peaks, each of which has thus been lettered here a, b, c and d in addition to the components numbered 1 to 15 described in the previous papers. Furthermore, the components 4 and 5, the separation of which had been rarely achieved with the previous carboxymethyl cellulose columns, were found to be separated with the CM52 columns (see Figs 10 to 13). (ii) Effect of cell-grinding conditions on the protein compositions of the isolated [30 S]- and 40 S nascent ribosomal particles It was found that labelled [30 s]- and 40 s nascent ribosomal particles isolated from exponentially growing cells by routine cell-grinding and fractionation procedures contained some non-ribosomal proteins which were situated between the components 1 and 4 on the chromatographs. On the other hand, no such proteins were found in [30 s]- and 40 s low CM-particles prepared by the routine procedures. These nonribosomal proteins could be those combined with 23 s rRNA of the respective nascent ribosomal particles during the extraction procedure, just as in the formation of socalled CM-particles in the cell extracts (Yoshida & Osawa, 1968). In the low CM-cells, the ratio of such non-ribosomal proteins to the particles was much less, compared with those in exponentially growing cells; only a small fraction of the particles would combine with the non-ribosomal proteins during the extraction procedure. It may then be expected that the combination of the non-ribosomal proteins to the particles in extracts of the exponentially growing cells may be reduced to a negligible extent by grinding the cells with an RNA preparation added in order to absorb the non-ribosomal proteins to it. The RNA used here was that prepared by the phenol method from cells which had been cultured in the presence of 400 pg CM/ml. for 1.5 hours, and is rich in nascent rRNA (Iwabuchi et al., 1965). The 40 s nascent ribosomal particles were prepared from cells homogeneously labelled with [3H]lysine with or without the RNA added during the cell grinding. The protein compositions of these two preparations were compared chromatographically. As expected, the former 40 s preparation (with the RNA added) had no detectable non-ribosomal proteins, while significant amounts of such proteins were detected in the latter (without the RNA added). It should be noted that the only change introduced by this addition of the RNA in the protein profile of the particles examined would concern the non-ribosomal proteins, since no difference in the protein profiles was found between 40 s low CMparticles prepared with and without the added RNA.

BIOSYNTHESIS

OF

50 B RIBOSOMES

337

(iii) Protein uwnpositiom of [30 Kj- and 40 S km chloramphenicol-particles Figure 10(a) and (b) illustrates the results of simultaneous chromatography on a CM52 column of [14C]lysine-labelled 50 s ribosomal protein and [3H]lysine-labelled [30 s]- (Fig. 10(a)) or 40 s (Fig. 10(b)) low CM-particles. The [30 s] low CM-particles contain only three main components, i.e. numbers 4, 6 and 10 out of 19 components recognizable in the 50 s ribosomal subunit by our method. The 40 s low CM-particles contain 12 components, i.e. 4,6,8,9, 10, 11, b, 12, 13, 14, 15 and c. The 40 slow CMparticles were found to include all the protein components present in the [30 s] low CM-particles. (iv) Protein compositions of [30 S]- and 40 S nascent ribosomal particles The chromatographic analyses of proteins of [30 s]- or 40 s nascent ribosomal particles prepared from the cells pulse-labelled with a mixture of [3H]amino acids for 200 seconds revealed that their protein compositions could not be distinguished from those of [30 s]- (Fig. 11) or 40 s low CM-particles. Similar protein analyses were performed on the 40 s nascent ribosomal particles from cells homogeneously labelled with r3H]amino acids for three generations. As seen in Figure 12, the protein compositions of the 40 s particles are indistinguishable from those of the 40 s low CMparticles (cf. Fig. 10(a)). The analysis of [30 s]-protein from the homogeneously labelled cells could not be conducted, since the ratio of the labelled [30 s] nascent ribosomal particles to the labelled 30 s ribosomal subunits was too small to obtain the former with a reasonable purity. (v) Protein compositions of lithium chloride-particles It has been reported that 40 s LXXparticles obtained by exposing 50 s ribosomal subunits to a concentrated LiCl solution have protein compositions very similar to those of the 40 s low CM-particles (Otaka et al., 1967 ; Itoh et al., 1968). The LiCl-particles lack at least the components 1, 2, 3 and either 4 or 5. Since the separation between number 4 and number 5 components could not be achieved with our previous carboxymethyl cellulose columns, it remains for us to decide which component, number 4 or 5, is missing in the 40 s LiCl-particles. Thus using a CM52 column, [14C]lysine-labelled protein of the 40 s LiCl-particles was analysed together with [3H]lysine-labelled 50 s ribosomal protein as reference. The result shown in Figure 13 clearly demonstrates the lack of number 4 component. The 40 s LiCl-particles are thus lacking six components, i.e. 1, 2, 3, a, 4 and 7. (d) Distribution

of 5 S RNA in various subribosomd particles and ribosomal subunits

The [30 s]- and 40 s low CM-particles and 50 s ribosomal subunits were prepared from the low CM-cells labelled with [3H]uridine for 20 minutes. RNA was then isolated from the respective particles and the distribution of 5 s RNA was examined by Sephadex 0100 column chromatography. As can be seen in Figure 14(a), (b) and (c), and Table 1, the 50 s ribosomal subunit contained approximately one molecule of 5 s RNA, assuming that molecular weights of 23 s rRNA and 5 s RNA are 1.1 x lo6 (Kurland, 1960) and 3.5~ lo4 (Brownlee, Sanger & Barrell, 1967), respectively. The 5 s RNA content in the preparations of [30 s]- or 40 s low CM-particles was less than 30?, of that in the preparation of the 50 s ribosomal subunit. 24

338

S. OSAWA,

E. OTAKA,

T. ITOH

AND

T. FUKUI

The analyses of 5 s RNA in the 40 s nascent ribosomel particles, and 50 s ribosomal subunit prepared from the cells labelled with 32P for three generations revealed the same situation; the 50 s ribosomal subunit contained about one molecule of 5 s RNA, while the 5 s content in the 40 s nascent ribosomal particle was at most 30% of that found in the 50 s ribosomal subunit (Fig. 15 and Table 1). The labelled 5 s RNA content in the 40 s nascent ribosomal particles from the cells pulse-labelled for 300 seconds with [3H]uridine was comparable with that of the 40 s particles from low CM- or homogeneously labelled cells, while that in the 50 s ribosomal subunit from the pulse-labelled cells was approximately 30% lower than that in the 50 s ribosomal subunit from low CM- or homogeneously labelled cells (Table 1). This probably means a delayed incorporation of [3H]uri&ne into 5 s RNA as compared with that into 23 s rRNA. [3H]Uridine-labelled RNA was also prepared from 30 s ribosomal subunit derived from low CM-cells, and 32P-labelled RNA from 30 s ribosomal subunit derived from homogeneously labelled cells. They were analysed for 5 s RNA content. No significant amount of 5 s was found in the above RNA preparations analysed. It may be thus concluded that, in confirmation of the results of Rosset, Monier & Julien (1964), Brown & Littna (1966), Comb & Zehavi-Willner (1967) and Knight & Darnell (1967), 5 s RNA is a constituent of the 50 s ribosomal subunit, and that at most only 30% of each class of isolated subribosomal particles contains 5 s RNA. During the preparation of this manuscript, the paper of Morel1 & Marmur (1968) was brought to our attention. They found that the 40 s nascent ribosomal particles do not contain an appreciable amount of 5 s RNA. The 5 s RNA content in the 40 s LiCl-particles was found to be approximately 30% of that in 50 s ribosomal subunit (Fig. 16 and Table l), indicating that the bulk 5 s RNA was removed from the subunit during the LiCl-treatment. (e) Methylation

of 23 S rRNA from various subribosomal particles and 50 s ribosomal subunit

An exponentially growing culture received [methyl-14C]methionine and [3H]uridine simultaneously (1) in the presence of O-8 pg CM/ml. (low CM-cells), (2) in its absence for 20 minutes, or (3) for 200 seconds. Under these conditions, the incorporation of 14C and 3H into trichloroacetic acid-precipitable materials occurred immediately after the addition of the isotopes and proceeded linearly at least during the above experimental periods. Thus the ratio of 3H to 14Cin RNA from the above three kinds of cells would not be influenced by the pools. The 40 s particles and 50 s ribosomal subunit were obtained as described in Materials and Methods and RNA was extracted from these particles. The [30 s]-particles were not purified. Instead, the RNA was directly extracted from the 30 s region after the first sucrose-gradient centrifugation to obtain 23 s rRNA from [30 s]-particles, since in this region the [30 s]-particles are the only component that contains 23 s rRNA. These RNA’s were again centrifuged in

Fm. 10. Chromatographic analyses on CM52 columns of protein components of [30 s]- and 40 s low CM-particles. [14C]Lysine-labelled 60 s protein was added aa a reference before analysis. (a) Protein of [30 s]low CM-particles and (b) protein of 40 s low CM-particles. --O--O--, Protein of 50 s ribosomal subunit (I%); -a--m-, protein of [30 s]- or 40 slow CM-particles (3H).

-V ,--(r---G=-CL-----am 12

-----+-XI ----*---d a%---------~----L -------0b --WC& % -0’ A axk=~-7 ----

-*wc&v---“---------I-----Q* - a- __________-^ - -- -- -- --- -- -- - - - - _- -- -0 - _ _ _ _ _

(z-01x u!~/siJ) HE

d

” <

-t= ar_-O-CI-+-- --4----.O--0-

Gr::- -4 _ 0 ---------o----------CL----

Ycb---Q-------_-_-___ pa:---------ta:-------------o----------o-

---______ _____

___ ___

~mQ----o-----o-----.

--+ -+

___-----_-__-----a--o----_----

--

cz-Ol

X “!~,‘*9J)Ht

ma--‘---‘--

5 4-D

-ne”‘-+---- ----ca-------

-------Q---8------L--O--

-NC --f-o,---

-1 43

(~-01

< #’

_----

*------‘-------’ w------------I WY

4

---..-w I P x U!WW)3

& PI

Cl

_ c

Labelling

TABLET

t 100 x (incorporation int,o 5 s;incorporation subunit (a)). molar ratios for the “2P-uniform $ The “true” might be influenced to some extent by possible

[‘4C]Adenine-labelled 50 s subunit exposed to 1.5 M-LiCl-0.1 M-Mg2 +

label ; interpretation pools, rates of synthesis,

given etc.

(a)

in the text

23 s + 5 s): (3aP.incorporation

40 s LiCl-particle

50 s ribosomal subunit 40 s low CM-particle [30 s]-low CM-particle

Cells labellad with [3H]uridine presence of 0.8 pg CM/ml.

in the for 20 min

50 s ribosomal subunit 40 s nascent ribosomal particle

into

from

60 s ribosomal subunit 40 s nascent ribosomal particle

prepared

Exponentially growing cells pulse-lab&xl with [3H]uridine for 300 set

cells labelled with for 3 generations

RNA

label

0.35

I.36 0.24 0.14

0.76 0.26

s)

of molar

rat I(,

5 s + 23 s of .;‘I s in terms

into

(5 s/23

0.313

I-14$

ratio

incorporation

Molar

by the ratio of 5s RNA

20 min-pulse

(a)fTthe 300 set- and

5 s of 50 s subunit on both

into

30.0

108 20.5 11.6

63.5 21.5

27.5

100

% of6sRNA.t

in 50 S ribosomal subunit, [30 S]- and 40 S subribosomd particles as determined to 23 s rRNA labelled with isotopes

conditions

of 5 s RNA

Exponentially growing [3aP]orthophosphate

Distribution

BIOSYNTHESIS

25

3s

40

45

25

30

OF

35

50 s RIBOSOMES

40

45

Fraction

no

50

343

25

30

35

!b)

i
40

45 O

(cl

FICL 14. Chromatographic analyses on Sephadex GlOO columns of RNA from low CM-particles and ribosomal subunits. Exponentially growing cells were incubated in 50 ml. of medium containing 0.8 pg CM/ml. and 500 PC of [3H]uridine and 250 pg of non-labelled uridine for 20 min. The [30 s]- and 40 s low CMparticles and 50 s ribosomal subunits were prepared by the method described under Materials and Methods. The RNA from the respective particles was mixed with a [‘Wladenine-labelled 5 s RNA preparation and fractionated on Sephadex GlOO columns. (a) RNA from [30 s] low CMparticles; (b) from 40 s low CM-particles; (c) from 50 s ribosomal subunit. -a-a--, RNA of the particles (3H); -O---O-, IO-fold magnification of the scale for sH; ------, 5 s RNA reference (IW).

45

30 Fraction

(a)

(b)

35

40

45

50

no

Cc)

FIG. 15. Chromatographic analyses on Sephadex Cl00 columns of RNA from 40 s nascent ribosomal particles and ribosomal subunits prepared from exponentially growing cells homogeneously lebelled with 3aP. Exponentially growing cells (50 ml.) were labelled with [3aP]orthophosphata (60 pc/ml. culture) for 3 generations. The 40 s nascent ribosomal particles and 50 s and 30 s ribosomal subunits were prepared as described in Materials and Methods. The RNA from the respective particles was fractionated on columns of Sephadex GIOO. (a) RNA from 40 s nascent ribosomal particles; (b) from 50 s ribosomal subunit; (c) from 30 s ribosomal subunit. --O-O-, IO-fold magnification of the scale.

344

S. OSAWA,

E. OTAKA,

T. ITOH

Fraction

AND

T. FUKUI

no.

Fm. 16. Chromatographic analysis on a Sephadex GlOO column of RNA from 40 s LiCl-particles obtained by exposing [14C]adenine-labelled 50 s ribosomal subunits to a concentrated LiCl solution.

4

5

IO

z h4 x

3

6

2

4

1

2

8

2 $3 Y I - 2

6

4

1

0

2

IO

20 (a)

0 30

0

IO Tub:b;o*

20

0 30 (cl

FIO. 17. Sucrose gradient centrifugation of 23 s rRNA of low CM-particles doubly labelled with [3H]uridine and [naetA@%]methionine. Exponentially growing cells (50 ml.) received [3H]uridine (100 PC) and [ntetAyZ-14C]methionine (30 pa) for 20 min in the presence of 0.8 pg CM/ml. Crude extracts were prepared from the cells and centrifuged in sucrose gradients in an SW25.1 rotor. RNA was prepared from material in the 60 s, 40 s and 30 s regions and examined by sucrose-gradient centrifugation. (a) 23 s rRNA from [30 s]-low CM-particles; (b) from 40 s low CM-particles; (c) from 50 s ribosomal subunit. -a-@-, 3H; --(J-m- 0-e. 14C.

sucrose gradients to separate 23 s rRNA from 16 s rRNA (Fig. 17) and the ratio of 3H to 14Cof each 23 s rRNA was estimated. As shown in Figure 17 and Table 2, the extents of methylation of 23 s rRNA component of the [30 s]- and 40 s particles from the above three types of cells were all about 60% of the 23 s rRNA derived from mature 60 s ribosomal subunit.

BIOSYNTHESIS

OF

60 s RIBOSOMES

345

4. Discussion (a) Identity

of [30 S]- or 40 S low chloramphenicol-particles with the [30 S]- or 40 S nascent ribosornal particles

It has been proposed that the intermediary steps of 50 s ribosomal subunit formation involve two precursor stages, namely, [30 S] (30 to 32 S) and 40 s (40 to 43 S) (Roberts et al., 1963; Mangiarotti et al., 1968; Osawa et al., 1967). These nascent ribosomal particles can easily be detected by pulse-labelling of the growing cells with isotopic precursors. When exponentially growing cells are treated with low concentrations of chloramphenicol, one finds in the cell extracts a considerable amount of low CM-particles having sedimentation coefficients and component RNA (23 s rRNA) indistinguishable from those of the [30 s]- and 40 s nascent ribosomal particles from the growing cells. In addition to the above characteristics, their protein compositions, 5 s RNA contents and methyl group contents in 23 s rRNA are about the same. It is however possible that the nascent ribosomal particles may still differ from the low CM-particles by one or more protein components which could not be resolved on the CM52 columns. In spite of this, we tentatively assume that the low CM-particles are synonymous with the nascent ribosomal particles. (b) On the existence of low chloramphenicol-

or nascent ribosomal particles

within the cells

It is rather difficult to obtain conclusive proof of the existence of low CM- (or nascent ribosomal) particles as such within the cells. There is usually more than one interpretation of the data derived from experiments of the type described in this paper, and the interpretations have certain limitations. We have given evidence indicating that low CM-rRNA is not mixed with high CM-rRNA in the course of harvesting the cells, the breakage of the cells or the preparation of cell extracts. This is consistent with the idea that low CM-particles are not the same kind of artifact as high CM-particles. However, it does not give us conclusive proof for the existence of low CM-particles in the cells, and only shows that the state of low CM-rRNA is different from that of high CM-rRNR in the cells. The following possibilities may be considered. (i) The lack of competition in the experiments cited above could result from a difference in the degree of maturation (e.g. of methylation) of low- and high CM-rRNA, which limits their ability to pick up proteins during the isolation procedures. (ii) Low CM-rRNA could be already bound to some protein in the cells. This could favour the “artificial” uptake of more proteins. (iii) Low CMparticles could bind more protein in the cells; during the isolation procedure some protein would be selectively released. (iv) Nascent ribosomal particles and low CMparticles could be those which have been derived from “newly formed ribosomal subunit,s” as a result of a release of some of their ribosomal proteins during the isolation procedures or even wit,hin the cells. The first possibility is not very likely, since it has been shown that low- and high CM-rRNA could not be distinguished from each other by their degree of methylation and their sedimentation properties; that is, the methyl content of “16 s” rRNA from both low- and high CM-particles was between 15 and 20% of the 16 s rRNA from mature 30 s subunit and that of 23 s rRNA from both particles was approximately 600/, of the 23 s rRNA from 50 s subunit (Hayashi, Osawa & Miura, 1966; Dubin & Giinalp, 1967 ; see also the results shown in this paper and a forthcoming paper). Moreover, all the submethylated rRNA described above sedimented as 17 s and 23 s instead of 16 s and 23 s for mature rRNA (Kono, Otaka &

2

t 100 x (‘%-incorporation 23 s rRNA of 50 s subunit).

into 23 s rRNA/3H-incorporation

into 23 s rRNA):

(‘Y-incorporation

into 23 s rRNA

of 50 s subunit/3H-incorporation

100 62 56

growing cells labelled with [methyl-14C] and [sH]uridine for 20 min

50 s ribosomal subunit 40 s low CM-particle [30 s]-low CM-particle

Exponentially methionine

Cells labelled with [methyZ-14C]methionine and r3H] uridine in the presence of 0.8 pg CM/ml. for 20 min

Methylation of 23 s rRNA (%)t

100 61 61

from:

50 s ribosomal subunit 40 s nascent ribosomal particle [30 s]-nascent ribosomal particle

prepared

Exponentially growing cells pulse-labelled with [methyllW]methionine and [3H]uridine for 200 seo

23 s rRNA

100 63 not examined

conditions

into

as determined by the

50 s ribosomal subunit 40 s nascent ribosomal particle [30 s]-nascent ribosomal particle

Labelling

Methyl group contents of 23 s rRNA prepared from 50 s ribosomal subunit, [30 S]- and 40 S subribosomal particles, ratio of 14C to 3H in the RNA labelled with [methyl-yC]methionine and [3H]uridine

TABLE

BIOSYNTHESIS

OF

50 s RIBOSOMES

347

Osawa, 1964; lwabuchi et al., 1965 ; and a forthcoming paper). A possibility that these rRNA’s are still different from each other in some other properties cannot be excluded however. The second and third possibilities, will now be discussed. In the experiments described in Results section( the labelled proteins of low CM-particles in the extract have been shown to be transferred to mature 50 s and 30 s ribosomal subunits upon removal of chloramphenicol in the presence of non-labelled lysine. Furthermore, no appreciable ribosomctl protein pool is present in the soluble protein fraction in the extract. If low CM-rRNA gains (additional) ribosomal proteins from the free protein pool (if present) during the extraction procedures, at least the bulk of such free ribosomal proteins should combine with low CM-rRNA to form low CM-particles, since no ribosomal protein was detected in the soluble fraction. Now, if there is any such hypothetical labelled free ribosomal protein pool within low CM-cells, it would be diluted with non-labelled free ribosomal proteins formed after the removal of chloramphenicol and in the presence of non-labelled lysine, provided that the rate of such dilution is sufficiently more rapid than that of 50 s and 30 s ribosome formation. As a result, the quantity of labelled ribosomal protein in the 50 s and 30 s subunits formed would be smaller than that of the labelled-protein in the original low CMparticles (in the extract). But it is not the case. On the other hand, if low CM-particles lose some ribosomal proteins during the fractionation procedures, the released proteins should exist in the soluble protein fraction. No such proteins were found however. The fourth possibility is also unlikely, since in the case of 50 s ribosome formation there exists a precursor-product relationship between 32 s (=[30 s]) and 43 s (=40 s) nascent ribosomal particles or between 43 s and 50 s ribosomal subunits (Mangiarotti et al., 1968), and RNA and protein components of the low CM-particles can be transferred to 50 s and 30 s ribosomal subunits. Furthermore, all nascent ribosomal particles and low CM-particles contain, unlike 50 s and 30 s ribosomal subunits, submethylated nascent rRNA. Thus all of the facts described above would suggest, though do not finally prove, that low CM-particles exist as such in the cells and can be transferred to the ribosomal subunits. The discussion on the biosynthesis of 50 s ribosomal subunit in this paper is based exclusively on the analyses of the isolated low CM- (or nascent ribosomal) particles, and therefore has obvious limitations which have been pointed out above. (c) Assembly

of ribosomal proteins

during

the formation

of 50 S ribosomal subunit

The analyses of protein compositions indicate that [30 s]-nascent ribosomal particles contain three main protein components, i.e. numbers 4, 6 and 10 (group I protein), and that 40 s nascent ribosomal particles have, in addition to group I protein, components 8, 9, 11,12,13,14,15, b and c (group II protein), total 12 out of 19 components detectable in the 50 s ribosomal subunit. These results may be interpreted as follows. In the early step of 50 s ribosomal subunit formation, group I protein is associated with 23 s rRNA, leading to the formation of [30 s]-nascent ribosomal particles. In the next step, group II protein is incorporated into the [30 s]-particles t,o make 40 s nascent ribosomal particle. In the final step, seven protein components (1, 2, 3, 5, 7, a and d; group III protein) are added to the 40 s particles. The number of protein components that are incorporated at each step cannot be accurate at present until more precise fractionation and characterization of each component are carried out. However, the important aspects here would be that the assembly of

348

S. OSAWA,

E. OTAKA,

T. ITOH

AND

T.

FUKUI

protein components is not a random process. It seems to proceed in stepwise fashion, certain specific groups of protein being added at each step. The preparation of [30 s]- (or 40 S) nascent ribosomal particles always contains a small amount of group II (or group III) protein (Figs 10 to 12). The reason for this is not known, but it might be that the [30 s]- (or 40 s) preparations contain a small amount of more “loosened” or “unfolded” 40 s nascent ribosomal particles (or 50 s ribosomal subunits) which behave like [30 s]- (or 40 s) particles in sucrose-gradient centrifugation or Sepharose 4B chromatography. This would account for small amounts of group II (or group III) protein on the CM52 chromatograms. This possibility is not unlikely, since sucrose-gradient centrifugation of dilute samples in a solution containing 10m4 M-i&‘+ of 50 s ribosomal subunits sometimes produces a small amount of 40 s materials which carry the original ratio of protein to RNA. An alternative explanation for the presence of minor amounts of group II (or group III) proteins and of 5 s RNA (see below) in the preparations of [30 s]- (or 40 s) particles is that the assembly of 50 s ribosomal subunit is not a strictly ordered process, and the [30 s]- and 40 s peaks each represents a heterogeneous population of particles having different compositions but similar sedimentation properties. This point should be clarified by further experiments. (d) Insertion

of 5 S RNA

during

the formation

of 50

S

ribosomal

subunit

The content of 5 s RNA in the preparations of [30 s]- and 40 s nascent ribosomal particles is only between 10 and 30% of 50 s ribosomal subunit. This suggests that the bulk of 5 s RNA is inserted during the transformation of 40 s nascent ribosomal particles to 50 s ribosomal subunits. A small amount of 5 s RNA in the 40 s preparations could be interpreted to mean that a certain population of the 40 s nascent ribosomal particles possesses 5 s RNA. However, it is also possible, as has been discussed in the case of protein composition, that the preparations contain a small amount of unfolded 50 s ribosomal subunit carrying 5 s RNA which sediments together with the 40 s nascent ribosomal particles. (Similar interpretations might be applicable to the protein compositions and 5 s RNA contents of 40 s LiCl-particles.) (e) Methyl&ion of 23 S rRNA during the formation of 50 S ribosomal subunii The contents of methyl groups in 23 s rRNA from [30 s]- and 40 s nascent ribosomal particles have been shown to be about 60% of mature 23 s rRNA from 50 s ribosomal subunit. These facts indicate that 6074 of methyl groups have been inserted into 23 s rRNA molecules until [30 s]-nascent ribosomal particles are formed, and the remainder of 40% is incorporated during the conversion of the 40 s nascent ribosomal particle to the 50 s ribosomal subunit, or immediately after the completion of the subunit. The significance of the late methylation of 23 s rRNA molecules at the end of the 50 s ribosome maturation is still obscure. One can suppose, for example, that the structural change due to the methylation of 23 s rRNA molecules might be responsible for the proper binding of proteins to the last intermediate to finish the ribosome formation, and/or this might be connected with the development of proper quaternary structure of ribosomes. In this respect, there is a suggestive paper by McDonald, Turnock $ Forchhammer (1966), who isolated a mutant strain of E. coli in which the ability to support protein synthesis of 70 s ribosomes is greatly impaired. They suggest

BIOSYNTHESIS

OF

349

50 s RIBOSOMES

that this abnormal property of ribosomes lies either in altered methylation of rRNA at the final step of the formation of 50 s subunit, or in the alteration of one of the structural proteins of the 50 s subunit resulting in abnormal quaternary structure of 50 s subunit. , 40s

sl

+ [30

+ 50 s subunit

1

-Formation

of 23 s rRNA

Early methyl&ion of 23 srRNA ... . . . . ... .. .. .. .. . .... ... Insertion of group I protein [components 4, 8 and 10 (3 components)]

.. ..... . .

.

.

.

I

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Insertion of group II protein [components 8,9,11, 12,13,14,15, b and c (9 camp.)]

.

.

. ...... . .. . ..

Late methyl&ion of 23 s rRNA ... .... . ... ... .. ... ... ... .. .. .. ... ... . .. ... . .. Insertion of group III protein [components 1, 2, 3, 5, 7, a and d (7 components)] I . .. . .. ... .. .. . Insertion

of 5 s RNA

FIQ. 18. Scheme for biosynthesis

of 50 s ribosomal

subunit in E. COG.

(f) Schemefor the formation of 50 S ribosomul subunit Considering all the data reported in this paper, the formation of 50 s ribosomal subunit may be summarized as follows. The first step involves the synthesis of 23 s rRNA, early methylation of 23 s rRNA and the combination of group I protein to the 23 s rRNA, leading to the formation of [30 s]-nascent ribosomal particles. The second step is the addition of group II protein to the [30 s]-nascent ribosomal particles, resulting in40 s nascent ribosomalparticles. The last step includes three kinds of event, namely, the attachment of group III protein to the 40 s nascent ribosomal particle, insertion of 6 s RNA and the late methylation of 23 s rRNA, thus leading to the completion of 50 s ribosomal subunit (Fig. 18). The above scheme has been constructed mainly from the data of molecular compositions of isolated nascent ribosomal particles. As already pointed out we are at present unable to exclude a possibility that each class of nascent particles contains more (or less) protein components or more (or less) 5 s RNA as it exists in the cells; during the isolation procedures, some of the protein or 5 s RNA could be lost from (or attached to) the particle. (g) Cmparison

of protein compositions of nascent ribosomul particles and lithium chloride particles

Lerman, Spirin, Gavrilova & Golov (1966) and Itoh et al. (1968) found that the detachment of protein from ribosomes with high concentrations of CsCl or LiCl proceeds stepwise by way of discrete stages; the salt-treatment of the 50 s (or 30 s) ribosomal subunit thus produces various protein-deficient particles whose sedimentation coeflicients are similar to those of nascent ribosomal particles. It has been also pointed out that the protein components of 40 s nascent ribosomal particles are similar to those of 40 s LiCl-particles (Otaka et al., 1967). In order to know the relation between protein-deficient particles obtained with salt treatment and nascent ribosomal

ribosomal

particles

particles

no.

~

-

1

-

~

2

-

-

3

+

+ -

4

-

-

/

5

+ +

+

-;A

6

+7 Present in full amount. -, Absent (components present less than 30% of the full amount & , Present in reduced amount. n.e., Not examined. t From Itoh et al., 1968.

36 s LiCl-partiolet 28 8 LiCl-p&i&t

[30 s]-nascent

40 s nascent ribosomal 40 s LiCl-particle

component

3

+ f

‘t

+

8

9

+ -

T

-+

were included

-

-

-

7

i-

’ ‘T

+

11

in this ~1~s).

+ *

z

+

10

+ +

+ -

+

12

f +

z

-+

13

* -

?

+-

14

Comparison of molecular compositions of nascent ribosomal particles and lithium

TABLE

f -

;

+

15

me. n.e.

1

-

a

n.e. n.e.

n.e. n.o.

+ + __

:. -

c or

b

chloride particles

-

n.e. me.

f_

d

-

1

-

5 s RNA

BIOSYNTHESIS

OF

50 s RIBOSOMES

351

particles, we have compared the protein compositions of nascent ribosomal particles with those of a series of LiCl-particles. The compositions are really very similar, though not exactly the same. The nascent 40 s lacks components 1,2,3, a, 5,7 and d, while components 1, 2, 3, a, 4 and 7 are missing in the LiCl-40 s particles (Table 3). Similar comparisons between [30 s]-nascent ribosomal particles with LiCl-36 s or 28 s particles also reveal certain definite differences (Table 3). For example, nascent [30 s] possesses the number 4 component, which is however missing in the LiCIparticles, etc. This work was supported in for Medical Research (project Japan.

part by a grant from the Jane Coffin Childs Memorial no. 233) and a grant

from

the Ministry

of Education

Fund of

REFERENCES Brown, D. D. & Littna, E. (1966). J. Mol. Biol. 20, 95. Brownlee, G. G., Sanger, F. & Barrell, B. G. (1967). Nuture, 215, 735. Comb, D. G. & Zehavi-Willner, T. (1967). J. Mol. Biol. 23, 441. Dubin, D. & Gunalp, A. (1967). Biochim. biophys. Acta, 134, 106. Galibert, F., Larsen, C. J., Lelong, J. C. & Boiron, M. (1965). Nature, 207, 1039. Galibert, F., Lelong, J. C., Larsen, C. J. & Boiron, M. (1967). Biochim. biophys.Acta, 142,89. Hayashi, Y., Osawa, S. & Miura, K. (1966). Biochim. biophys. Acta, 129, 519. Itoh, T., Otaka, E. & Osawa, S. (1968). J. Mol. BioZ. 33, 109. Iwabuchi, M., Kono, M., Oumi, T. & Osawa, S. (1965). Biochim. biophys. Acta, 108, 211. Knight, E. Jr. & Darnell, J. E. (1967). J. Mol. BioZ. 28, 491. Kono, M., Otaka, E. & Osawa, S. (1964). Biochim. biophys. Acta, 91, 612. Kurland, C. G. (1960). J. Mol. BioZ. 2, 93. Leive, L. (1965). J. Mol. BioZ. 13, 862. Lerman, M. L., Spirin, A. S., Gavrilova, L. P. & Golov, V. F. (1966). J. Mol. BioZ. 15, 268. Mangiarotti, G., Apirion, D., Schlessinger, D. & Silengo, L. (1968). Biochemistry, 7, 456. McDonald, R., Turnock, G. & Forchhammer, J. (1966). Proc. Nat. Acad. Sci., Wash. 57, 141. Morell, P. & Marmur, J. (1968). Biochemistry, 7, 1141. Morell, P., Smith, J., Dubanau, D. & Marmur, J. (1967). Biochemistry, 6, 268. Muto, A., Otaka, E. & Osawa, S. (1966). J. Mol. BioZ. 19, 60. Osawa, S., Otaka, E., Muto, A., Yoshida, K. & Itoh, T. (1967). Proc. 7th Int. Congr. Biothem. Abstracts I, p. 119. Tokyo: Science Council of Japan. Otaka, E., Itoh, T. & Osawa, S. (1967). Science, 157, 1452. Otaka, E., Itoh, T. & Osawa, S. (1968). J. Mol. BioZ. 33, 93. Roberts, R. B., B&ten, R. J. & McCarthy, B. J. (1963). In Molecular Genetics, ed. by J. H. Taylor, part I, p. 291. New York: Academic Press. Rosset, R., Monier, R. & Julien, J. (1964). BUZZ. Sot. Chim. BioZ. 46, 87. Watanabe, I. (1957). J. Gen. Physiol. 40, 521. Yoshida, K. & Osawa, S. (1968). J. Mol. BioZ. 33, 559.