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Cyclic dissociation into stable subunits and re-formation of ribosomes during bacterial growth
J. Mol. Biol. (1968) 31, 277-289
Cyclic Dissociation into Stable Subunits and Re-formation of Ribosomes during Bacterial Growth RAYI\IOND 0. R. KAEIVJPFER,MATTHEW
NESELSOW AND HESCIIEL
,J. RASK~S~
The Biological Laboratories Harrard Cnivemity Cambridge, Massachusetts, U.S.A. (Received 7 September 1967) Studies of the distribution of isotopic labels among ribosomes and ribosomal subunits following transfer of a growing bacterial culture from a heavy- to a lightisotopes medium show that: (1) During growth ribosomes frequently undergo subunit exchange, presumably by dissociation into their 30 s and 50 s subunits and re-formation from a pool of free subunits. (2) Ribosomal subunits are stable: they remain intact during growth and are continuously. recycled through ribosomes. On the basis of these results, it is suggested that ribosomes must dissociate into their subunits between successive rounds of translation.
1. Introduction Throughout nature, ribosomes are found to be composed of two subunits, one approximately twice the size of the other (for review see Schweet & Heintz, 1966). In bacteria, the ribosomes active in protein synthesis have a sedimentation coefficient value of 70 s; they consist of two subunits sedimenting at 30 s and 50 s, respectively. According to the generally accept,ed view of prot)ein synthesis (for review see Watson, 1964), ribosomes move unidirectionally along a messenger RNA molecule as its nucleotide sequence is translated into the peptide sequence of a growing polypeptide chain. After the synthesis of one or more polypeptides encoded on the same messenger, t)he ribosome is released from t’he messenger RNA and becomes available for further rounds of translation. The reason for the universal bipartit’e construction of ribosomes is unknown. In particular, it is not known if the existence of two separate subunits serves some purpose in protein synthesis and, if so, what that might be. In this paper, we ask if the 30 s and 50 s subunits of ribosomes are permanently associated in vice or if, instead, ribosomes undergo subunit exchange during normal growt’h. To answer this question we have examined the density distribution of ribosomes in extracts made following the transfer of growing bacteria uniformly labeled with heavy isotopes to a medium containing only light isotopes. To interpret the distribution, we needed to know if the 30 s and 50 s subunits synthesized before transferremain heavy during growth in light medium. Density analysis of the subunits following transfer shows that they do, and that they remain free of ribosomal protein synthesized after transfer. + Present address: St’. Louis, RIO, U.S.A.
Institute
for Molecular
Virology, 277
St. Louis
‘University
Medical
School
278
R. 0. R. KAEMPFER,
M. MESELSON
AND
H. J. RASKAS
At the time of transfer, all ribosomes are heavy. If they undergo subunit exchange during growth in light medium, heavy ribosomes should be progressively replaced by two species of hybrid densit.y. One of the hybrids should contain a heavy 50 s and a light 30 s subunit, the other a light 50 s and a heavy 30 s subunit. Our finding is that during growth in light medium, heavy ribosomes do indeed disappear while hybrid species are formed. We conclude that 30 s and 50 s ribosomal subunits are stable during normal bacterial growth and that ribosomes undergo subunit exchange, presumably in connection with their role in protein synthesis.
2. Materials and Methods (a) Bacterial strains and media The uracil-requiring strain Escherichia coli 13148 was used for transfer experiments and the RNase I-deficient strain DlO (Gesteland, 1966) was used as carrier. Heavy isotopes medium consisted of D,O with 0.08 M-Tris (pH 7.4), 0.01 &I-NaCl, 0.005 M-MgSO,, 6 x low6 M-FeCl,, 10m6 M-call,, 0.01 M-‘~NH&~, 25 PC/ml. [3H]uracil, and sufficient [13C, 15N, DIalgal hydrolysate (Meselson & Weigle, 1961) to support the growth of 5 x lo8 cells/ml. Light isotopes medium consisted of H,O with 0.08 al-Tris (pH 7.4), 0.006 ivr-NaCl, 0.005 M-M~SO~, 3 x lOwe m-FeCl,, 0.25% glycerol, O.lo/, yeast extract, 0.15% Casamino acids (0.015% in the experiment illustrated in Fig. 2), 10 pg/ml. of each of the four RNA nucleosides and 10 pg/ml. uracil. The light isotopes medium was conditioned before use by growing strain B148 to 2 x lo7 cells/ml. and removing the cells by centrifugation and Millipore filtration.
(b) Transfer experiments Bacteria were grown at 37°C in heavy medium for 10 generations. Growth was followed by cell counting in a Petroff-Hausser chamber. When the cells reached a titer of approximately 108/ml., unlabeled uridine (17.5 pg/ml.) was added. 25 min later, the cells (1.5 to 1.8 x 108/ml.) were sedimented by centrifuging at room temperature for 4 min, washed with a small amount of warm light medium, and resuspended at 2 x lo7 cells/ml. in light medium at 37°C. Approximately 9 min elapsed between harvesting and resuspension. When growth was to be continued beyond 3.5 generations following transfer, additional warm light medium was added. Samples were withdrawn at intervals, rapidly mixed with 0.1 vol. of frozenTrisbuffer (0.01 M-Tris (pH 7.4), 0.01 M-magnesium acetate, 0*0005n1EDTA) and made 0.015 M in NaN,. (c) Preparation of cell extracts All operations were carried out near 0°C. The cells were centrifuged, washed once and resuspended in Tris buffer. When mixed extracts were to be analyzed, the cells were mixed at this stage. Unless otherwise’ noted, frozen exponential-phase carrier cells were added to an excess of at least 20-fold. The cells were then centrifuged in a small glass tube. To the pellet were added 10 ~1. each of Tris buffer and 1% gelatin, 10 ,ug DNase, and sufficient levigated alumina to obtain a paste ; the cells were then ground in the tube with a Teflon plunger for about 1 min. The paste was extracted with 0.4 ml. Tris buffer and RNase was added to 0.05 pg/ml. For sucrose gradient sedimentation, but not for CsCl density-gradient analysis, KC1 was added to 9 mg/ml. and sodium deoxycholate to 5 me/ ml. After centrifugation for 15 min at low speed, the extracts were immediately subjected to ultracentrifugation. The extracts contained more than 70% of the radioactivity incorporated by the cells.
(d) Sedimentation analysis Enzy-me-grade sucrose (Mann Research Laboratories, Inc.) was used to prepare 12.5 to 19.6% exponential gradients. Low-magnesium gradients contained 0.01 M-sodium phosphate buffer, pH 7.0, 0.001 M-magnesium acetate, 0.05 ~-Kc1 and 100 pg/ml. gelatin; they were cenbrifuged at 5°C for 4.0 hr in the SB283 rotor of the International ultracentri-
CYCLIC
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279
fuge at 41,000 rev./min. High-magnesium gradients contained 0.01 M-Tris buffer, pH 7.4, O*Ol M-magnesium acetate, 0.05 M-KCl, and 100 rg/ml. gelatin. They were centrifuged for 3.5 hr under the same conditions. Drops were collected in tubes, or on filter paper squares which were dried and counted in a liquid-scintillation spectrometer. (e) Density-gradient centrifugation .4 0.2-ml. vol. of cell extract was rapidly mixed in a thin-walled polypropylene centrifuge tube with 3.8 ml. cesium chloride solution at pH 7.3, containing 0.01 M-Tris buffer and 0.05 M-magnesium acetate, with a refractive index 7t5 = l-3975. The polypropylene tubes had been boiled twice in 0.1 M-NasCO,, 0.005 M-EDTA, several times in water, s,nd finally once in 0.01 M-magnesium acetate followed by a water rinse. The samples were centrifuged for 18 hr at 45,000 rev./min at 5°C in the SB405 rotor of the International ultracentrifuge. Drops were collected in tubes containing 0.25 ml. 0.01 M-Tris (pH 7.4), 0.01 M-magnesium acetate, 100 rg/ml. gelstin at O”C, or directly onto filter paper discs. Equal portions from the tubes were spotted on filter discs, processed as described by Bollum (1959), and counted in a liquid-scintillation spectrometer. The combined use of carrier, Mg2 + -treated tubes and short time of centrifugation may be necessary to ensure the stability of ribosomes in CsCl. More than 90% of the input radioactive material was recovered from sucrose and cesium chloride gradients.
3. Results (a) Bacterial
growth in isotope transfer
experiments
Figure 1 illustrates the course of growth following transfer of bacteria from [‘“C, 15N, D] to pre-conditioned [12C,14N, HImedium. It can be seen that in these circum-
Time
(hr)
FIQ. 1. Cell growth during an isotope transfer experiment. The cell titers in light medium (0) are multiplied by 9 to correct for dilution. The doubling time before and after transfer is 42 min. ( l ), Cell titers in heavy medium.
280
R. 0. R. KAEMPFER,
M. MESELSON
AND
H. J. RASKAS
stances growth of the cells proceeds unabated throughout the isotope transfer experiment. Following transfer from heavy to light medium, the ribosome number, estimated from the uptake of 32P, initially increases at a rate slightly faster than the cell number, in keeping with the observation t,hat the cells become larger. (b) Conservation of ribosomul subunits It was found by Meselson, Nomura, Brenner, Davern & Schlessinger (1964) that 30 s and 50 s ribosomal subunit,s lose about 20% of their protein when banded in CsCl, becoming particles called cores. They also found in heavy isotope transfer experiments that no more than 5% per generation of the RNA or protein of ribosomal cores is exchanged during exponential growth. This result, however, does not tell whether the CsCl-dissociable protein of the subunits exchanges in viva. A means to study exchange in the intact ribosomal subunits was provided by the observation that on sucrose gradients heavy subunits sediment faster than their light counterparts. This allows the combined density and size analysis of ribosomal particles in a single gradient.
Fraction
number (b)
(a)
FIG. 2. Conservation
of 50 s subunits
during exponential
growth.
Cells were labeled with [3H]uracil in heavy medium and transferred to light medium contwiuing [‘%]amino acids. Growth was continued for 1.3 generations after transfer. 5 min before harvestillg, ]‘2C]amino acids (100 mg,‘ml.) were added. Washed cells were lysed by brief sonication in 0.01 RIsodium phosphate buffer, pH 7.0, 0.001 sr-Mg2+, 10 pg/ml. DSasc in the ahseucr of carrier cells. (a) Sedinwntation distribution of 30 s and 50 s subunits in 0.001 II-Q’ +. (I)) Rqion -4 (contninmg the heavy 50 s subunits) was dialyzed against 0.01 nr-sodium phosphate buffw (pH 7.0). 0.001 h1.nIg2+,0.03 wKC1, and re-examined in 0.001 M-M$+. Rackground was not subtracted from t’he 14C counts. -.-a--, 3H; -O-O-, 14C.
CYCLIC
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281
The separation of heavy and light 30 s and 50 s subunits is shown in Fig. 2(a). The RNA of the heavy ribosomes was labeled with [3H]uracil; the protein synthesized after transfer from heavy to light medium n-as labeled with [l”C]amino acids. The cells were harvested after l-3 generations in light medium. The amount of 14C label under the heavy 50 s peak provides a measure of protein exchange in the heavy 50 s subunits during growth in light medium. Comparison of Fig. 2(a) and (b) shows that no more than 1% of the l*C in 50 s subunits is in heavy 50 s. This corresponds to no more than 3% equilibrated between the heavy and light 50 s populations. IVe
IO F Heavy
Light: -
IO
20
30
40
50
Fraction
number
60
70
80
90
f-Y----
+3
CYCLIC
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OF RIBOSOMES
283
conclude that 50 s subunits exchange less than 3% of their protein in I-3 generat’ions. Evidence for the conservation of the 30 s subunit will be presented below. (c) Density separation of ribosomes by sucrose gradient sedimentation A sucrose gradient analysis of a mixture containing heavy and light ribosomes and subunits is illustrated in Fig. 3. All species are well resolved: the heavy particles sediment more than 20% faster than the corresponding light ones. Note that aggregation between heavy and light species does not 0ccur.t This technique provides an assay for subunit exchange in vivo: following transfer from heavy to light medium, such exchange should result in the accumulation of hybrid ribosomes sedimenting in the region intermediate between 86 s and 70 s. (d) Subunit exchnge in ribosomes : sedimentation velocity a,nalysis A portion of the heavy, [3H]uracil-containing culture used to prepare the extract analyzed in Fig. 3 was transferred to light medium. Analysis of extracts made before and immediately after transfer showed identical density distributions. Three and a half generations after transfer (Fig. 4(a) and (b)) the radioactivity initially associated with heavy ribosomes is found in a position intermediate between heavy (86 s) and light (70 s), although individual hybrid species are not resolved. At the time of harvest,ing, the cells should contain approximately a tenfold excess of light ribosomes. If subunit exchange occurs in a time short compared to the bacterial generation time, and if all ribosomes are subject to subunit exchange, there should be approximately ten times more hybrid ribosomes than all-heavy ones. The predominance of hybrid over heavy ribosomes seen in Pig. 4(a) and (b) is consistent with this expectation. Figure 4(b) reveals that after 3.5 generations of growth in light meuium the heavy 30 s and 50 s subunits sediment at exactly the same positions as the original heavy subunits. Exchange of approximately 2% of the material of the 50 s subunit or 4p:, of the 30 s subunit would have resulted in an observable density displacement. Therefore, both ribosomal subunits are completely or almost completely conscrl-et1 during growth. Figure 4(b) further shows that the proportion of free heavy subunits does not increase after transfer: during growth there appears to be no accumulation of subunits incapable of re-entering ribosomes. (e) Subunit exchange ins ribosomes: CsCl density-gradient analysis Since the two expected hybrid ribosome species were not resolved on sucrose gradients, an attempt was made to detect them in CsCl gradients at sedimentation t It may be noted that the ratio of heavy 60 s to 30 s free subunits is slightly larger than that observed for the light subunits. This result, consistently seen also when subunit,s derived from the 66 s peal; arc analyzed, may indicate that heavy 30 s subunits are less efficiently recovered during preparation.
FIG. 4. Sedimentation
distribution of heavy ribosomes heavy to light medium.
following
transfer
of cells from
to unlabeled light medium and Cells grown in honvy, r3H] uracil medium were transferred growth was continued for 3.5 generations. Prior to extraction, [‘4C]uracil-lnbelod light cells (n j or [‘Wlurncil-lnbeled heavy ~011s (b) were added to provide a reference. The indicated positions of heavy 70 s ribosomes in (a), and of light 70 s ribosomes ilk (b) are derived from the gradient in Fig. 3, which was run simultaneously. Centrifugation was cerried out in 0.01 ar-Mg2 +. -•--a---, 3H; --o--o-, 1%.
284
R. 0. R. KAEMPFER,
M. MESELSON
AND
Heavy !
H. J. RASKAS
Light r”
“r
Fraction
r”
number
FIN. 5. Cesium chloride density-gradient ana.lyses of extracts made at 0.1 and 1.5 generations after transfer of bacteria from heavy, [sH]uracil medium to 32P-containing light medium. Two separate gradients are presented : one contains ribosomes from cells harvested 5 min after transfer (--A--A--, 3H); the other from cells harvested after 1.5 generations. Both gradients contain exactly the same amounts of carrier ribosomes and approximately equal numbers of ribosomes from the transfer experiment. A total of 50 fractions was collected per gradient. Peak assignments (arrows) are based on refractive index measurements. -O--e--, 3H; -O-O-, =P.
equilibrium. Brenner, Jacob $ Meselson (1961) found that, at a high Mg2+ concentration, ribosomes yield two bands in CsCl. The denser A band contains proteindeficient cores of the 30 s and 50 s subunits, while ribosomes are found only in the lighter B band (Meselson et al., 1964).t At low ribosome concentrations, the B band tends to be converted to A band, but under our conditions a substantial fraction of the ribosomes is in the B form. t There a,re indicat’ions that the B band may also contain (T. Stachelin, personal communication).
some intact
ribosomal
subunits
CYCLIC
DISSOCIATION
285
OF RIBOSOMES
Cells were labeled with [3H]uracil in heavy medium and transferred to light medium containing 32P0,. The density distribution of the control sample (0.1 generation transfer) seen in Fig. 5 displays approximatjely equal amounts of A and B band. There is no evidence of aggregation of heavy mat,erial with the light carrier ribosomes. As may be seen in Fig. 5, at 1.5 generations after transfer the amount of all-hea.vy B band ribosomes has greatly decreased, concomitant with the appearance of ribosomes of hybrid density containing 3H and 32P label. The near disappearance of the fully heavy B band suggests that subunit exchange keeps pace with the synthesis of light subunits, and agrees with the evidence from sucrose gradients (Fig. 4) that the average time a pair of subunits stays together is not more than a cell generation, and possibly is much less. Since in the zero-time sample no 3H label is found at hybrid density, and since, as we have shown, ribosomal subunits are conserved, it follows that the hybrid material found after transfer must consist of complexes containing both heavy and light subunit,s. Presumably, these complexes are 70 s ribosomes, although other species may also be present (see below).
2
1
0
20
30
40
0 20 Fraction
30
40
20
30
40
number
FIG. 6. Sedimentation distribution of ribosomal subunits derived from ribosomcs with hybrid densities. Regions P, Q and R from the gradient presented in Fig. 5 were dialyzed against O-01 wsodium phosphate buffer, pH 7.0, 0.001 ix-Mg 2+, 0.05 M-KC1 in the presence of purified carrier ribosomes and examined by sucrose gradient centrifugation in 0.001 n~-Mg2+. Note that the scales are different in the three graphs. -.-a--, 3H; -O-O--, =P.
286
R. 0. R. KAEMPFER,
M. YESELSON
AND
H. J. RASKAS
Subunit exohange in ribosomes should result in the appearance of two hybrid B bands, equally spaced between the heavy and light B band positions. The heavier hybrid peak should contain ribosomes consisting of a heavy 50 s and a light 30 s subunit and vice veraa for the lighter peak. The hybrid regions P and & of the gradient shown in Fig. 5 were analyzed on low-magnesium sucrose gradients. Figure 6 shows that the ribosomes of region P consist, as expected, of heavy 50 s and light 30 s subunits, with only very few light 50 s and heavy 30 s particles. Analysis of region Q shows the presence of heavy 60 s subunits, an unexpected result. Particles banding at this position must contain heavy and light isotopes in a ratio considerably leas than one. Therefore, the presence of heavy 50 s particles implies that single ribosomes cannot be the only species present. This is also suggested by the observation that the amount of 3H label in region Q is more than half the amount in region P. Some of the particles in region Q may contain two ribosomes, with one of the 50 s subunit8 heavy. Since the control shows that aggregation does not take place during extraction and analysis, such dimers, if they exist, must have originated in viva. Perhaps they are dimera or disomes which escaped the action of RNase during extraction. The problem is being studied. The expected lighter hybrid (heavy 30 s and light 50 s) may well be present in region Q, since there are substantial numbers of heavy 30 s and light 50 s subunits. Region R (light B band), serving as control, is seen to contain light 30 s and 50 s subunits in equal numbers. It may be noted in Fig. 5 that the light B band predominates over the light A band. The high concentration of carrier ribosomes present in this region of the gradient may be acting to stabilize the light B band. Beyond 1.5 generations after transfer, there is relatively little change in the density distribution of heavy label. Figure 7 shows the distribution at 3.2 generations. Little if any all-heavy B band is seen, in agreement with the evidence from sucrose gradients that all ribosomes are subject to exchange. Comparison of Figs 5 and 7 shows essentially no change in the shape of the hybrid area. The hybrid region was found to retain its shape even five generations after transfer, as expected if a majority of the hybrid particles are simply single ribosomes. In particular, the fact that peak P did not diminish noticeably between 1.5 and 5 generations after transfer constitutes strong evidence that the material in this region indeed consists of single ribosomes. Any more complex aggregates would at such late times contain a proportion of heavy isotopes too small to be found in region P. 4. Discussion Two basic features of ribosome metabolism emerge from the experiments presented here: (1) During growth ribosomes frequently undergo subunit exchange, presumably by dissociation into their 30 s and 50 s subunits and re-formation from a pool of free subunits. (2) Ribosomal subunits are stable: they remain intact during growth and are continuously recycled through ribosomes. Our evidence for these conclusions derives from studies of the distribution of isotopic labels among ribosomes and subunits following transfer of a growing bacterial culture from a heavy to a light isotopes medium. The distributions were analyzed by two different techniques: sedimentation velocity analysis on sucrose gradients and equilibrium density-gradient centrifugation in CsCl solution, The limitations and
CYCLIC
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287
--T-----l-----r-rHeavy
Light 3
1 ?
1
Fraction
--I40
number
FIG. 7. C&urn chloride density-gradient analyses of extracts made at 0.1 and 3-2 generations after transfer of bacteria from heavy, [3B]uracil medium to 32P-containing light medium. Sea the legend to Fig. 6.
complementary characteristics of these techniques have been pointed out in Results. The conservation of 30 s and 50 s ribosomal subunits during bacterial growth was shown by the absence of detectable protein exchange in heavy 50 s subunits and by the observation that heavy subunits retain their original density during growth in light medium. Subunit exchange in ribosomes was shown by the fact that growth in light medium results in the conversion of heavy ribosomes to hybrid density. All or most of the ribosomes were found to undergo exchange within a time no longer than a cellular generation and possibly much less. There was no evidence for the existence of ribosomes which could not dissociate, nor for the accumulation of 30 s or 60 s subunits unable to cycle through ribosomes.
288
R. 0. R. KAEMPFER,
M. MESELSON
AND H. J. RASKAS
The simplest explanation for subunit exchange is that it constitutes a mandatory phase in protein synthesis : Upon leaving a messenger RNA molecule at the completion of translation, the ribosome dissociates into its two subunits. The subunits join an intracellular pool from which ribosomes are re-formed at the initiation of a new round of translation. Although subunit exchange may occur only after several cycles of translation, there is no a priori reason to assume this. The simplest explanation of our results is that subunit exchange in ribosomes occurs after every round of translation, that is, every 10 to 30 seconds (McQuillen, Roberts t Britten, 1959; Schleif, 1967). This is a time much shorter than can be resolved by our experiments. The cyclic dissociation and re-formation of ribosomes has been proposed by other authors on the basis of several lines of evidence (Mangiarotti & Schlessinger, 1966, 1967; Nomura & Lowry, 1967). Mangiarotti & Schlessinger (1966) find that E. coli extracts prepared by supposedly gentle methods contain polysomes and free subunits, but no single ribosomes. They suggest that single ribosomes do not exist in vivo, consistent with the cyclic model described above. Single ribosomes were, however, found in similar studies by Flessel, Ralph & Rich (1967), Kelley & Schaechter (manuscript in preparation) and Kohler, Ron & Davis (manuscript in preparation). In an attempt to obtain more direct evidence for subunit exchange and to measure its rate, Mangiarotti & Schlessinger (1967) compared the specific activity of the free subunits in extracts of pulse-labeled cells to that of the subunits derived from polysomes. They interpret the results in terms of rapid and extensive exchange. It should be pointed out, however, that this interpretation requires that the subunits studied by Mangiarotti & Schlessinger are not derived from single ribosomes or polysomes during extraction. The postulated necessity of subunit exchange during protein synthesis might serve a regulatory function. If subunits can associate to form active ribosomes only at the starting points for translation on the messenger RNA, this would provide a safeguard against spurious initiation of translation of the messages of the cell. Recent in vitro studies may be pertinent to this hypothesis. The first step in the assembly of ribosomes from subunits appears to be the association of the 30 s subunit with the initiator sequence AUG on messenger RNA and the formylmethionyltransfer RNA specific for the AUG codon (Nomura & Lowry, 1967). This view is in accord with the observation that the in vitro formation of ribosomes from native subunits requires messenger RNA and transfer RNA, and that messenger RNA containing the sequence AUG is particularly effective (Schlessinger, Mangiarotti 6 Apirion, 1967). In vitro, single ribosomes, but not 30 s subunits, can bind to numerous codons other than the initiator (Nomura &, Lowry, 1967). Such binding of free ribosomes to non-initiating codons would not occur in vivo if ribosomes dissociate after translation. Subunit exchange in ribosomes has not been demonstrated in animal cells; however, it appears that free subunits are the only ribosomal particles active in the initiation of hemoglobin synthesis in rabbit retioulocytes (Bishop, 1966a$). In HeLa cells, newly synthesized vaccinia virus messenger RNA is associated with the small ribosomal subunit before it appears in polysomes (Joklik C%Becker, 1965). Thus, it seems likely that animal ribosomes also undergo subunit exchange.
CYCLIC
DISSOCIATION
OF RIBOSOMES
This work was supported by grants from the National Jane CofKn Childs Memorial Fund for Medical Research. Fellow of the latter organization.
289
Science Foundation and the One of us (R. 0. R. 9.) is a
Note added in proof. Recently we have found that a small proportion, about 5%, of the particles in extracts prepared for CsCl centrifugation sediment as disomes on sucrose gradients. These complexes persist in O-05 M-KCl, but are converted to single ribosomes upon incubation with 5 pg ribonuclease/ml. for 5 min at 37°C. The CsCl gradient distribution of transferred ribosomes completely freed of disomes is not yet established.
REFERENCES biophys. Acta, 119, 130. Bishop, J. 0. (1966a). &o&m. Bishop, J. 0. (19665). J. Mol. Biol. 17, 285. Bollum, F. S. (1959). J. Biol. Chem. 234, 2733. Brenner, S., Jacob, F. & Meselson, M. (1961). Nature, 190, 576. -’ Flessel, C. P., Ralph, P. & Rich, A. (1967). Science, 1.58, 658. Gesteland, R,. F. (1966). J. Mol. BioZ. 17, 67. Joklik, W. K. & Becker, Y. (1965). J. Mol. BioZ. 13, 511. Mangiarotti, G. & Schlessinger, D. (1966). J. Mol. BioZ. 20, 123. Mangiarotti, G. & Schlassinger, D. (1967). J. Mol. BioZ. 29, 395. McQuillen, K., Roberts, R. B. & Britten, R. J. (1959). Proc. Nat. Ad.
Sci.,
Wmh.
45,
1437.
Meselson, M., Nomura,
M., Brenner,
S., Davern,
C. & Schlessinger,
D. (1964). J. MOE.
BioZ. 9, 696.
Meselson, M. & Weigle, J. J. (1961). Proc. Nat. Ad. Sci., Wash. 47, 857. Nomura, M. & Lowry, C. V. (1967). Proc. Nut. Acad. Sci., Wash. 58, 946. Schleif, R. (1967). J. Mol. BioZ. 27, 41. Schlessinger, D., Mangiarotti, F. & Apirion, D. (1967). Proc. Nut. Ad. Sci., Wash. 58, 1782. Schweet,, R. & Heintz, R. (1966). Ann. Rev. Biochem. 35, 723. Watson, J. D. (1964). Bull. Sot. Chim. biol. 12, 1399.
Cyclic Dissociation into Stable Subunits and Re-formation of Ribosomes during Bacterial Growth RAYI\IOND 0. R. KAEIVJPFER,MATTHEW
NESELSOW AND HESCIIEL
,J. RASK~S~
The Biological Laboratories Harrard Cnivemity Cambridge, Massachusetts, U.S.A. (Received 7 September 1967) Studies of the distribution of isotopic labels among ribosomes and ribosomal subunits following transfer of a growing bacterial culture from a heavy- to a lightisotopes medium show that: (1) During growth ribosomes frequently undergo subunit exchange, presumably by dissociation into their 30 s and 50 s subunits and re-formation from a pool of free subunits. (2) Ribosomal subunits are stable: they remain intact during growth and are continuously. recycled through ribosomes. On the basis of these results, it is suggested that ribosomes must dissociate into their subunits between successive rounds of translation.
1. Introduction Throughout nature, ribosomes are found to be composed of two subunits, one approximately twice the size of the other (for review see Schweet & Heintz, 1966). In bacteria, the ribosomes active in protein synthesis have a sedimentation coefficient value of 70 s; they consist of two subunits sedimenting at 30 s and 50 s, respectively. According to the generally accept,ed view of prot)ein synthesis (for review see Watson, 1964), ribosomes move unidirectionally along a messenger RNA molecule as its nucleotide sequence is translated into the peptide sequence of a growing polypeptide chain. After the synthesis of one or more polypeptides encoded on the same messenger, t)he ribosome is released from t’he messenger RNA and becomes available for further rounds of translation. The reason for the universal bipartit’e construction of ribosomes is unknown. In particular, it is not known if the existence of two separate subunits serves some purpose in protein synthesis and, if so, what that might be. In this paper, we ask if the 30 s and 50 s subunits of ribosomes are permanently associated in vice or if, instead, ribosomes undergo subunit exchange during normal growt’h. To answer this question we have examined the density distribution of ribosomes in extracts made following the transfer of growing bacteria uniformly labeled with heavy isotopes to a medium containing only light isotopes. To interpret the distribution, we needed to know if the 30 s and 50 s subunits synthesized before transferremain heavy during growth in light medium. Density analysis of the subunits following transfer shows that they do, and that they remain free of ribosomal protein synthesized after transfer. + Present address: St’. Louis, RIO, U.S.A.
Institute
for Molecular
Virology, 277
St. Louis
‘University
Medical
School
278
R. 0. R. KAEMPFER,
M. MESELSON
AND
H. J. RASKAS
At the time of transfer, all ribosomes are heavy. If they undergo subunit exchange during growth in light medium, heavy ribosomes should be progressively replaced by two species of hybrid densit.y. One of the hybrids should contain a heavy 50 s and a light 30 s subunit, the other a light 50 s and a heavy 30 s subunit. Our finding is that during growth in light medium, heavy ribosomes do indeed disappear while hybrid species are formed. We conclude that 30 s and 50 s ribosomal subunits are stable during normal bacterial growth and that ribosomes undergo subunit exchange, presumably in connection with their role in protein synthesis.
2. Materials and Methods (a) Bacterial strains and media The uracil-requiring strain Escherichia coli 13148 was used for transfer experiments and the RNase I-deficient strain DlO (Gesteland, 1966) was used as carrier. Heavy isotopes medium consisted of D,O with 0.08 M-Tris (pH 7.4), 0.01 &I-NaCl, 0.005 M-MgSO,, 6 x low6 M-FeCl,, 10m6 M-call,, 0.01 M-‘~NH&~, 25 PC/ml. [3H]uracil, and sufficient [13C, 15N, DIalgal hydrolysate (Meselson & Weigle, 1961) to support the growth of 5 x lo8 cells/ml. Light isotopes medium consisted of H,O with 0.08 al-Tris (pH 7.4), 0.006 ivr-NaCl, 0.005 M-M~SO~, 3 x lOwe m-FeCl,, 0.25% glycerol, O.lo/, yeast extract, 0.15% Casamino acids (0.015% in the experiment illustrated in Fig. 2), 10 pg/ml. of each of the four RNA nucleosides and 10 pg/ml. uracil. The light isotopes medium was conditioned before use by growing strain B148 to 2 x lo7 cells/ml. and removing the cells by centrifugation and Millipore filtration.
(b) Transfer experiments Bacteria were grown at 37°C in heavy medium for 10 generations. Growth was followed by cell counting in a Petroff-Hausser chamber. When the cells reached a titer of approximately 108/ml., unlabeled uridine (17.5 pg/ml.) was added. 25 min later, the cells (1.5 to 1.8 x 108/ml.) were sedimented by centrifuging at room temperature for 4 min, washed with a small amount of warm light medium, and resuspended at 2 x lo7 cells/ml. in light medium at 37°C. Approximately 9 min elapsed between harvesting and resuspension. When growth was to be continued beyond 3.5 generations following transfer, additional warm light medium was added. Samples were withdrawn at intervals, rapidly mixed with 0.1 vol. of frozenTrisbuffer (0.01 M-Tris (pH 7.4), 0.01 M-magnesium acetate, 0*0005n1EDTA) and made 0.015 M in NaN,. (c) Preparation of cell extracts All operations were carried out near 0°C. The cells were centrifuged, washed once and resuspended in Tris buffer. When mixed extracts were to be analyzed, the cells were mixed at this stage. Unless otherwise’ noted, frozen exponential-phase carrier cells were added to an excess of at least 20-fold. The cells were then centrifuged in a small glass tube. To the pellet were added 10 ~1. each of Tris buffer and 1% gelatin, 10 ,ug DNase, and sufficient levigated alumina to obtain a paste ; the cells were then ground in the tube with a Teflon plunger for about 1 min. The paste was extracted with 0.4 ml. Tris buffer and RNase was added to 0.05 pg/ml. For sucrose gradient sedimentation, but not for CsCl density-gradient analysis, KC1 was added to 9 mg/ml. and sodium deoxycholate to 5 me/ ml. After centrifugation for 15 min at low speed, the extracts were immediately subjected to ultracentrifugation. The extracts contained more than 70% of the radioactivity incorporated by the cells.
(d) Sedimentation analysis Enzy-me-grade sucrose (Mann Research Laboratories, Inc.) was used to prepare 12.5 to 19.6% exponential gradients. Low-magnesium gradients contained 0.01 M-sodium phosphate buffer, pH 7.0, 0.001 M-magnesium acetate, 0.05 ~-Kc1 and 100 pg/ml. gelatin; they were cenbrifuged at 5°C for 4.0 hr in the SB283 rotor of the International ultracentri-
CYCLIC
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279
fuge at 41,000 rev./min. High-magnesium gradients contained 0.01 M-Tris buffer, pH 7.4, O*Ol M-magnesium acetate, 0.05 M-KCl, and 100 rg/ml. gelatin. They were centrifuged for 3.5 hr under the same conditions. Drops were collected in tubes, or on filter paper squares which were dried and counted in a liquid-scintillation spectrometer. (e) Density-gradient centrifugation .4 0.2-ml. vol. of cell extract was rapidly mixed in a thin-walled polypropylene centrifuge tube with 3.8 ml. cesium chloride solution at pH 7.3, containing 0.01 M-Tris buffer and 0.05 M-magnesium acetate, with a refractive index 7t5 = l-3975. The polypropylene tubes had been boiled twice in 0.1 M-NasCO,, 0.005 M-EDTA, several times in water, s,nd finally once in 0.01 M-magnesium acetate followed by a water rinse. The samples were centrifuged for 18 hr at 45,000 rev./min at 5°C in the SB405 rotor of the International ultracentrifuge. Drops were collected in tubes containing 0.25 ml. 0.01 M-Tris (pH 7.4), 0.01 M-magnesium acetate, 100 rg/ml. gelstin at O”C, or directly onto filter paper discs. Equal portions from the tubes were spotted on filter discs, processed as described by Bollum (1959), and counted in a liquid-scintillation spectrometer. The combined use of carrier, Mg2 + -treated tubes and short time of centrifugation may be necessary to ensure the stability of ribosomes in CsCl. More than 90% of the input radioactive material was recovered from sucrose and cesium chloride gradients.
3. Results (a) Bacterial
growth in isotope transfer
experiments
Figure 1 illustrates the course of growth following transfer of bacteria from [‘“C, 15N, D] to pre-conditioned [12C,14N, HImedium. It can be seen that in these circum-
Time
(hr)
FIQ. 1. Cell growth during an isotope transfer experiment. The cell titers in light medium (0) are multiplied by 9 to correct for dilution. The doubling time before and after transfer is 42 min. ( l ), Cell titers in heavy medium.
280
R. 0. R. KAEMPFER,
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AND
H. J. RASKAS
stances growth of the cells proceeds unabated throughout the isotope transfer experiment. Following transfer from heavy to light medium, the ribosome number, estimated from the uptake of 32P, initially increases at a rate slightly faster than the cell number, in keeping with the observation t,hat the cells become larger. (b) Conservation of ribosomul subunits It was found by Meselson, Nomura, Brenner, Davern & Schlessinger (1964) that 30 s and 50 s ribosomal subunit,s lose about 20% of their protein when banded in CsCl, becoming particles called cores. They also found in heavy isotope transfer experiments that no more than 5% per generation of the RNA or protein of ribosomal cores is exchanged during exponential growth. This result, however, does not tell whether the CsCl-dissociable protein of the subunits exchanges in viva. A means to study exchange in the intact ribosomal subunits was provided by the observation that on sucrose gradients heavy subunits sediment faster than their light counterparts. This allows the combined density and size analysis of ribosomal particles in a single gradient.
Fraction
number (b)
(a)
FIG. 2. Conservation
of 50 s subunits
during exponential
growth.
Cells were labeled with [3H]uracil in heavy medium and transferred to light medium contwiuing [‘%]amino acids. Growth was continued for 1.3 generations after transfer. 5 min before harvestillg, ]‘2C]amino acids (100 mg,‘ml.) were added. Washed cells were lysed by brief sonication in 0.01 RIsodium phosphate buffer, pH 7.0, 0.001 sr-Mg2+, 10 pg/ml. DSasc in the ahseucr of carrier cells. (a) Sedinwntation distribution of 30 s and 50 s subunits in 0.001 II-Q’ +. (I)) Rqion -4 (contninmg the heavy 50 s subunits) was dialyzed against 0.01 nr-sodium phosphate buffw (pH 7.0). 0.001 h1.nIg2+,0.03 wKC1, and re-examined in 0.001 M-M$+. Rackground was not subtracted from t’he 14C counts. -.-a--, 3H; -O-O-, 14C.
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281
The separation of heavy and light 30 s and 50 s subunits is shown in Fig. 2(a). The RNA of the heavy ribosomes was labeled with [3H]uracil; the protein synthesized after transfer from heavy to light medium n-as labeled with [l”C]amino acids. The cells were harvested after l-3 generations in light medium. The amount of 14C label under the heavy 50 s peak provides a measure of protein exchange in the heavy 50 s subunits during growth in light medium. Comparison of Fig. 2(a) and (b) shows that no more than 1% of the l*C in 50 s subunits is in heavy 50 s. This corresponds to no more than 3% equilibrated between the heavy and light 50 s populations. IVe
IO F Heavy
Light: -
IO
20
30
40
50
Fraction
number
60
70
80
90
f-Y----
+3
CYCLIC
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283
conclude that 50 s subunits exchange less than 3% of their protein in I-3 generat’ions. Evidence for the conservation of the 30 s subunit will be presented below. (c) Density separation of ribosomes by sucrose gradient sedimentation A sucrose gradient analysis of a mixture containing heavy and light ribosomes and subunits is illustrated in Fig. 3. All species are well resolved: the heavy particles sediment more than 20% faster than the corresponding light ones. Note that aggregation between heavy and light species does not 0ccur.t This technique provides an assay for subunit exchange in vivo: following transfer from heavy to light medium, such exchange should result in the accumulation of hybrid ribosomes sedimenting in the region intermediate between 86 s and 70 s. (d) Subunit exchnge in ribosomes : sedimentation velocity a,nalysis A portion of the heavy, [3H]uracil-containing culture used to prepare the extract analyzed in Fig. 3 was transferred to light medium. Analysis of extracts made before and immediately after transfer showed identical density distributions. Three and a half generations after transfer (Fig. 4(a) and (b)) the radioactivity initially associated with heavy ribosomes is found in a position intermediate between heavy (86 s) and light (70 s), although individual hybrid species are not resolved. At the time of harvest,ing, the cells should contain approximately a tenfold excess of light ribosomes. If subunit exchange occurs in a time short compared to the bacterial generation time, and if all ribosomes are subject to subunit exchange, there should be approximately ten times more hybrid ribosomes than all-heavy ones. The predominance of hybrid over heavy ribosomes seen in Pig. 4(a) and (b) is consistent with this expectation. Figure 4(b) reveals that after 3.5 generations of growth in light meuium the heavy 30 s and 50 s subunits sediment at exactly the same positions as the original heavy subunits. Exchange of approximately 2% of the material of the 50 s subunit or 4p:, of the 30 s subunit would have resulted in an observable density displacement. Therefore, both ribosomal subunits are completely or almost completely conscrl-et1 during growth. Figure 4(b) further shows that the proportion of free heavy subunits does not increase after transfer: during growth there appears to be no accumulation of subunits incapable of re-entering ribosomes. (e) Subunit exchange ins ribosomes: CsCl density-gradient analysis Since the two expected hybrid ribosome species were not resolved on sucrose gradients, an attempt was made to detect them in CsCl gradients at sedimentation t It may be noted that the ratio of heavy 60 s to 30 s free subunits is slightly larger than that observed for the light subunits. This result, consistently seen also when subunit,s derived from the 66 s peal; arc analyzed, may indicate that heavy 30 s subunits are less efficiently recovered during preparation.
FIG. 4. Sedimentation
distribution of heavy ribosomes heavy to light medium.
following
transfer
of cells from
to unlabeled light medium and Cells grown in honvy, r3H] uracil medium were transferred growth was continued for 3.5 generations. Prior to extraction, [‘4C]uracil-lnbelod light cells (n j or [‘Wlurncil-lnbeled heavy ~011s (b) were added to provide a reference. The indicated positions of heavy 70 s ribosomes in (a), and of light 70 s ribosomes ilk (b) are derived from the gradient in Fig. 3, which was run simultaneously. Centrifugation was cerried out in 0.01 ar-Mg2 +. -•--a---, 3H; --o--o-, 1%.
284
R. 0. R. KAEMPFER,
M. MESELSON
AND
Heavy !
H. J. RASKAS
Light r”
“r
Fraction
r”
number
FIN. 5. Cesium chloride density-gradient ana.lyses of extracts made at 0.1 and 1.5 generations after transfer of bacteria from heavy, [sH]uracil medium to 32P-containing light medium. Two separate gradients are presented : one contains ribosomes from cells harvested 5 min after transfer (--A--A--, 3H); the other from cells harvested after 1.5 generations. Both gradients contain exactly the same amounts of carrier ribosomes and approximately equal numbers of ribosomes from the transfer experiment. A total of 50 fractions was collected per gradient. Peak assignments (arrows) are based on refractive index measurements. -O--e--, 3H; -O-O-, =P.
equilibrium. Brenner, Jacob $ Meselson (1961) found that, at a high Mg2+ concentration, ribosomes yield two bands in CsCl. The denser A band contains proteindeficient cores of the 30 s and 50 s subunits, while ribosomes are found only in the lighter B band (Meselson et al., 1964).t At low ribosome concentrations, the B band tends to be converted to A band, but under our conditions a substantial fraction of the ribosomes is in the B form. t There a,re indicat’ions that the B band may also contain (T. Stachelin, personal communication).
some intact
ribosomal
subunits
CYCLIC
DISSOCIATION
285
OF RIBOSOMES
Cells were labeled with [3H]uracil in heavy medium and transferred to light medium containing 32P0,. The density distribution of the control sample (0.1 generation transfer) seen in Fig. 5 displays approximatjely equal amounts of A and B band. There is no evidence of aggregation of heavy mat,erial with the light carrier ribosomes. As may be seen in Fig. 5, at 1.5 generations after transfer the amount of all-hea.vy B band ribosomes has greatly decreased, concomitant with the appearance of ribosomes of hybrid density containing 3H and 32P label. The near disappearance of the fully heavy B band suggests that subunit exchange keeps pace with the synthesis of light subunits, and agrees with the evidence from sucrose gradients (Fig. 4) that the average time a pair of subunits stays together is not more than a cell generation, and possibly is much less. Since in the zero-time sample no 3H label is found at hybrid density, and since, as we have shown, ribosomal subunits are conserved, it follows that the hybrid material found after transfer must consist of complexes containing both heavy and light subunit,s. Presumably, these complexes are 70 s ribosomes, although other species may also be present (see below).
2
1
0
20
30
40
0 20 Fraction
30
40
20
30
40
number
FIG. 6. Sedimentation distribution of ribosomal subunits derived from ribosomcs with hybrid densities. Regions P, Q and R from the gradient presented in Fig. 5 were dialyzed against O-01 wsodium phosphate buffer, pH 7.0, 0.001 ix-Mg 2+, 0.05 M-KC1 in the presence of purified carrier ribosomes and examined by sucrose gradient centrifugation in 0.001 n~-Mg2+. Note that the scales are different in the three graphs. -.-a--, 3H; -O-O--, =P.
286
R. 0. R. KAEMPFER,
M. YESELSON
AND
H. J. RASKAS
Subunit exohange in ribosomes should result in the appearance of two hybrid B bands, equally spaced between the heavy and light B band positions. The heavier hybrid peak should contain ribosomes consisting of a heavy 50 s and a light 30 s subunit and vice veraa for the lighter peak. The hybrid regions P and & of the gradient shown in Fig. 5 were analyzed on low-magnesium sucrose gradients. Figure 6 shows that the ribosomes of region P consist, as expected, of heavy 50 s and light 30 s subunits, with only very few light 50 s and heavy 30 s particles. Analysis of region Q shows the presence of heavy 60 s subunits, an unexpected result. Particles banding at this position must contain heavy and light isotopes in a ratio considerably leas than one. Therefore, the presence of heavy 50 s particles implies that single ribosomes cannot be the only species present. This is also suggested by the observation that the amount of 3H label in region Q is more than half the amount in region P. Some of the particles in region Q may contain two ribosomes, with one of the 50 s subunit8 heavy. Since the control shows that aggregation does not take place during extraction and analysis, such dimers, if they exist, must have originated in viva. Perhaps they are dimera or disomes which escaped the action of RNase during extraction. The problem is being studied. The expected lighter hybrid (heavy 30 s and light 50 s) may well be present in region Q, since there are substantial numbers of heavy 30 s and light 50 s subunits. Region R (light B band), serving as control, is seen to contain light 30 s and 50 s subunits in equal numbers. It may be noted in Fig. 5 that the light B band predominates over the light A band. The high concentration of carrier ribosomes present in this region of the gradient may be acting to stabilize the light B band. Beyond 1.5 generations after transfer, there is relatively little change in the density distribution of heavy label. Figure 7 shows the distribution at 3.2 generations. Little if any all-heavy B band is seen, in agreement with the evidence from sucrose gradients that all ribosomes are subject to exchange. Comparison of Figs 5 and 7 shows essentially no change in the shape of the hybrid area. The hybrid region was found to retain its shape even five generations after transfer, as expected if a majority of the hybrid particles are simply single ribosomes. In particular, the fact that peak P did not diminish noticeably between 1.5 and 5 generations after transfer constitutes strong evidence that the material in this region indeed consists of single ribosomes. Any more complex aggregates would at such late times contain a proportion of heavy isotopes too small to be found in region P. 4. Discussion Two basic features of ribosome metabolism emerge from the experiments presented here: (1) During growth ribosomes frequently undergo subunit exchange, presumably by dissociation into their 30 s and 50 s subunits and re-formation from a pool of free subunits. (2) Ribosomal subunits are stable: they remain intact during growth and are continuously recycled through ribosomes. Our evidence for these conclusions derives from studies of the distribution of isotopic labels among ribosomes and subunits following transfer of a growing bacterial culture from a heavy to a light isotopes medium. The distributions were analyzed by two different techniques: sedimentation velocity analysis on sucrose gradients and equilibrium density-gradient centrifugation in CsCl solution, The limitations and
CYCLIC
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287
--T-----l-----r-rHeavy
Light 3
1 ?
1
Fraction
--I40
number
FIG. 7. C&urn chloride density-gradient analyses of extracts made at 0.1 and 3-2 generations after transfer of bacteria from heavy, [3B]uracil medium to 32P-containing light medium. Sea the legend to Fig. 6.
complementary characteristics of these techniques have been pointed out in Results. The conservation of 30 s and 50 s ribosomal subunits during bacterial growth was shown by the absence of detectable protein exchange in heavy 50 s subunits and by the observation that heavy subunits retain their original density during growth in light medium. Subunit exchange in ribosomes was shown by the fact that growth in light medium results in the conversion of heavy ribosomes to hybrid density. All or most of the ribosomes were found to undergo exchange within a time no longer than a cellular generation and possibly much less. There was no evidence for the existence of ribosomes which could not dissociate, nor for the accumulation of 30 s or 60 s subunits unable to cycle through ribosomes.
288
R. 0. R. KAEMPFER,
M. MESELSON
AND H. J. RASKAS
The simplest explanation for subunit exchange is that it constitutes a mandatory phase in protein synthesis : Upon leaving a messenger RNA molecule at the completion of translation, the ribosome dissociates into its two subunits. The subunits join an intracellular pool from which ribosomes are re-formed at the initiation of a new round of translation. Although subunit exchange may occur only after several cycles of translation, there is no a priori reason to assume this. The simplest explanation of our results is that subunit exchange in ribosomes occurs after every round of translation, that is, every 10 to 30 seconds (McQuillen, Roberts t Britten, 1959; Schleif, 1967). This is a time much shorter than can be resolved by our experiments. The cyclic dissociation and re-formation of ribosomes has been proposed by other authors on the basis of several lines of evidence (Mangiarotti & Schlessinger, 1966, 1967; Nomura & Lowry, 1967). Mangiarotti & Schlessinger (1966) find that E. coli extracts prepared by supposedly gentle methods contain polysomes and free subunits, but no single ribosomes. They suggest that single ribosomes do not exist in vivo, consistent with the cyclic model described above. Single ribosomes were, however, found in similar studies by Flessel, Ralph & Rich (1967), Kelley & Schaechter (manuscript in preparation) and Kohler, Ron & Davis (manuscript in preparation). In an attempt to obtain more direct evidence for subunit exchange and to measure its rate, Mangiarotti & Schlessinger (1967) compared the specific activity of the free subunits in extracts of pulse-labeled cells to that of the subunits derived from polysomes. They interpret the results in terms of rapid and extensive exchange. It should be pointed out, however, that this interpretation requires that the subunits studied by Mangiarotti & Schlessinger are not derived from single ribosomes or polysomes during extraction. The postulated necessity of subunit exchange during protein synthesis might serve a regulatory function. If subunits can associate to form active ribosomes only at the starting points for translation on the messenger RNA, this would provide a safeguard against spurious initiation of translation of the messages of the cell. Recent in vitro studies may be pertinent to this hypothesis. The first step in the assembly of ribosomes from subunits appears to be the association of the 30 s subunit with the initiator sequence AUG on messenger RNA and the formylmethionyltransfer RNA specific for the AUG codon (Nomura & Lowry, 1967). This view is in accord with the observation that the in vitro formation of ribosomes from native subunits requires messenger RNA and transfer RNA, and that messenger RNA containing the sequence AUG is particularly effective (Schlessinger, Mangiarotti 6 Apirion, 1967). In vitro, single ribosomes, but not 30 s subunits, can bind to numerous codons other than the initiator (Nomura &, Lowry, 1967). Such binding of free ribosomes to non-initiating codons would not occur in vivo if ribosomes dissociate after translation. Subunit exchange in ribosomes has not been demonstrated in animal cells; however, it appears that free subunits are the only ribosomal particles active in the initiation of hemoglobin synthesis in rabbit retioulocytes (Bishop, 1966a$). In HeLa cells, newly synthesized vaccinia virus messenger RNA is associated with the small ribosomal subunit before it appears in polysomes (Joklik C%Becker, 1965). Thus, it seems likely that animal ribosomes also undergo subunit exchange.
CYCLIC
DISSOCIATION
OF RIBOSOMES
This work was supported by grants from the National Jane CofKn Childs Memorial Fund for Medical Research. Fellow of the latter organization.
289
Science Foundation and the One of us (R. 0. R. 9.) is a
Note added in proof. Recently we have found that a small proportion, about 5%, of the particles in extracts prepared for CsCl centrifugation sediment as disomes on sucrose gradients. These complexes persist in O-05 M-KCl, but are converted to single ribosomes upon incubation with 5 pg ribonuclease/ml. for 5 min at 37°C. The CsCl gradient distribution of transferred ribosomes completely freed of disomes is not yet established.
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Meselson, M. & Weigle, J. J. (1961). Proc. Nat. Ad. Sci., Wash. 47, 857. Nomura, M. & Lowry, C. V. (1967). Proc. Nut. Acad. Sci., Wash. 58, 946. Schleif, R. (1967). J. Mol. BioZ. 27, 41. Schlessinger, D., Mangiarotti, F. & Apirion, D. (1967). Proc. Nut. Ad. Sci., Wash. 58, 1782. Schweet,, R. & Heintz, R. (1966). Ann. Rev. Biochem. 35, 723. Watson, J. D. (1964). Bull. Sot. Chim. biol. 12, 1399.