JOURNAL
OF
Vol. 126, No. 3 Printed in U.S.A.
BACTERIOLOGY, June 1976, p. 1149-1155
Copyright © 1976 American Society for Microbiology
Ribosomal Precursor Particles of Bacillus megaterium BARBARA A. BODY AND BERNARD H. BROWNSTEIN* Department of Cell Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40506
Received for publication 1 March 1976
Pulse-labeled cells ofBacillus megaterium were converted to protoplasts, and lysates of the protoplasts were analyzed by sucrose gradient sedimentation. Precursor ribonucleoprotein (RNP) particles then appeared predominantly as 50S and 30S precursor ribosomal subunits. Polyacrylamide gel electrophoresis of the ribosomal ribonucleic acid from the 50S and 30S RNP particles confirmed their precursor nature since they were shown to contain precursor 23S and 16S ribosomal ribonucleic acid, respectively. Treatment of protoplast lysates with 0.5% deoxycholate prior to sedimentation analysis resulted in a markedly different radioactivity profile. The 50S RNP particles were no longer present, but 43S particles were observed in addition to increased amounts of pulse-labeled material sedimenting at 30S and slower. Extracts from cells broken in a French press showed a profile from sucrose gradient sedimentation similar to that of the deoxycholate-treated protoplast lysate. These data suggest that the nature ofthe precursor ribosomal particles appears to be a function of the method of cell disruption or detergent treatment of the cell extract preparation. The observed 509 and 30S RNP particles may be the major precursor ribosomal subunits in vivo; the slower-sedimenting species could result from some form of breakdown or change in the configuration of the 50S and 30S precursors. Britten et al. (2, 10) first demonstrated the existence of precursor ribosomal subunits. Further work by Mangiarotti et al. (11) identified three ribosomal subunit precursors, two for the 50S subunit and one for the 30S subunit. Other studies of various mutants that accumulate ribonucleoprotein particles (RNP) corresponding to the classical ribosomal precursor stages (4-7, 9-11) contributed to the hypothesis that these precursors may correspond to ratelimiting steps in the subunit assembly process. Ribosome assembly has also been examined by characterizing in vitro reconstitution of subunits (12, 18, 24). An intermediate particle was observed when mature 16S ribosomal ribonucleic acid (rRNA) and 30S proteins were used for reconstitution of 30S ribosomal subunits. However, this intermediate has a somewhat different protein composition than that of the in vivo 30s precursor (15). Conversion of this particle to a functional 30S subunit was shown to be temperature dependent (24). Also, Mangiarotti et al. (12) successfully used precursor 16S rRNA in the reconstitution of active 30S subunits and showed that it is not temperature dependent and has no intermediate like that observed with mature rRNA. Thus, the in vivo assembly process seems somewhat different than that inferred from in vitro reconstitution systems.
Recently, two new ribosomal precursors that cosediment with the mature 50S and 30S subunits were reported by Lindahl (8, 9). The slower-sedimenting ribosomal precursors were also observed, but it was concluded that the 50S and 30S cosedimenting particles were the major precursor species and that they were not observed in other studies because of their relative instability. Variations in observed sedimentation values of classical precursor particles have been reported (6, 19) which could also be due to instability. In this study of ribosomal precursors ofBacillus megaterium we have also demonstrated that the major precursor particles cosediment with the mature 50S and 30S subunits. Furthermore, the sedimentation of ribosomal precursors observed was, in fact, dependent on the method of cell disruption employed. Vigorous and mild cell breakage techniques, as well as detergent treatments, have a profound effect on the properties of the ribosomal precursor particles. MATERIALS AND METHODS Bacterium and growth of cultures. The bacterium used throughout this study was B. megaterium KM. Stock cultures of the organism were maintained on slants of stock culture medium (Difco Laboratories). In all experiments the bacteria were
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BODY AND BROWNSTEIN
grown in the minimal medium of Wachsman and Storch (28) modified to contain 0.5% glucose and 20% (wt/vol) sucrose and supplemented with 40 ,ug of Larginine per ml. All cultures were grown at 30 C in a gyratory shaker that rotated at 250 rpm. They exhibited a 60-min generation time. GrQwth was monitored with a Klett-Summerson colorimeter using a no. 54 filter (540 nm); one Klett unit equaled 9 x 105 cells per ml. Isotopic labeling of cells. Flasks containing fresh medium were inoculated with a 1:100 dilution from an overnight culture. After one doubling, cells were steady-state labeled by the addition of 0.2 ,uCi of [2'4C]uracil (specific activity, 56 mCi/mmol) per ml to the culture. After four more generations, cells were pulse labeled for 2.5 min by the addition of 2 ,uCi of [5,6-3H]uridine (specific activity, 26 Ci/mmol) per ml. At the end of the 2.5-min pulse period, sodium azide and chloramphenicol were added to final concentrations of 1 mM and 100 yg/ml, respectively. Cells prepared in this manner were either converted to protoplasts or subjected to the treatments described below. Preparation of protoplasts and protoplast lysates. Protoplast formation was accomplished by the procedure of Tremblay et al. (25), with the following modifications. The sodium azide- and chloramphenicol-treated cells were immediately poured into a prewarmed Erlenmeyer flask that contained lysozyme (EC 3.2.1.17) such that the final concentration would be 200 ,ug/ml. The flask was then swirled in a 37 C water bath for 30 s. The suspension was then poured over a 4/5 volume of crushed, frozen TM2KS buffer [10 mM tris(hydroxymethyl)aminomethanehydrochloride, pH 7.2; 10 mM magnesium acetate; 100 mM KCl; 20% (wt/vol) sucrose]. All subsequent steps were carried out at 0 to 4 C. Protoplasts were harvested by centrifugation at 6,000 x g for 7 min in a Sorvall RC2-B centrifuge. The supernatant fluid was discarded. The pellet of protoplasts was suspended and lysed in cold TM4K buffer [10 mM
tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.2; 0.1 mM magnesium acetate; 50 mM KClI containing deoxyribonuclease (EC 3.1.4.5) at 10 ,ug/ ml. In those experiments in which an alternate technique was used to break protoplasts, the pellet was suspended in TM4K buffer containing 10% (wt/ vol) sucrose and immediately broken by the alternative technique. Preparation of whole-cell extracts by using a French pressure cell. Cells not converted to protoplasts were treated in a manner identical to that described above, except that lysozyme was omitted. Resuspended cells or protoplasts were broken by passage through a chilled French pressure cell at 10,000 lb/in2. The broken cell preparation was collected in a chilled tube containing deoxyribonuclease at 10 ,ig/ml. After protoplasts were broken by this procedure, the preparation was subsequently diluted by adding 3 volumes of TM4K buffer. All lysates and extracts were either used immediately or frozen and stored at -70 C. The data obtained from stored extracts did not differ significantly from those obtained from extracts used immediately. Detergent and lysozyme treatment of lysates.
J. BACTERIOL.
The portions of protoplast lysates or cell extracts that were to be detergent or lysozyme treated were incubated with sodium deoxycholate (DOC) at 0.5% or lysozyme at 200 ,ug/ml for 15 min at 0 C prior to sedimentation analysis. Sucrose gradient sedimentation analysis. Ribosomal subunits and precursor particles were separated by centrifugation of the protoplast lysate or cell extracts through linear 5 to 20% (wt/vol) sucrose-TM4 (TM4K without KCl) gradients. The gradients were formed over a 1.5-ml 80% (wt/vol) sucrose-TM4 cushion used to catch material that might otherwise form a pellet (21). A Beckman SW27 rotor with 17-ml tubes was used for all sedimentation analysis. After 10.7 h of centrifugation at 4 C, the tubes were punctured at the bottom with a needle in a drop-collecting apparatus to collect fractions. Usually 40 fractions were obtained per gradient. RNA analysis of lysates, extracts, and particles. The sucrose gradient fractions containing either the 50S or 30S subunits or precursor particles were individually pooled and subsequently precipitated by raising the magnesium ion concentration to 20 mM and adding 0.7 volume of cold ethanol. After 30 min at -20 C, the precipitates were collected by centrifugation at 12,000 x g for 30 min in the cold. The pellets were then suspended in ASES buffer (10 mM sodium acetate, pH 5.2; 100 mM sodium chloride; 1 mM disodium ethylenediaminetetraacetate [EDTA]; 0.2% sodium dodecyl sulfate [SDS]). When appropriate, the crude cell extracts and protoplast lysates were prepared for RNA analysis by the addition of 0.2% SDS (final concentration). Polyacrylamide gel electrophoresis. The method of Bishop et al. (1) was used for RNA analysis. The samples were layered on cylindrical gels (9 by 90 mm) and developed by regulating the current at 10 mA/gel in E buffer (1). The developed gels were frozen at - 40 C on aluminum trays prior to preparation for radioactive analysis. A Mickel gel slicer was used to obtain 0.5-mm slices, which were subsequently dried on GF/A paper (Reeve-Angel). The paper was then placed in scintillation vials and the radioactivity was determined as described below. Determination of radioactivity. The radioactivity of gradient fractions was measured by one of two methods. In the first method, 200 ,ug ofbovine serum albumin (Miles Laboratories; purified fraction V) was added to each fraction and then precipitated with cold 5% trichloroacetic acid. The precipitates were collected on GF/A filters and washed three times with cold 5% trichloroacetic acid. They were then dried, put into glass scintillation vials containing toluene-Omnifluor scintillation fluid (New England Nuclear Corp.), and counted in a Packard TriCarb liquid scintillation spectrometer, model 3375. The second method was utilized only when fractions were also to be used for RNA analysis. In this case, a 20-,Al sample from each fraction was spotted on a GF/C filter and placed into 5% trichloroacetic acid. The filters were rinsed with three changes of 5% trichloroacetic acid followed by three rinses with acetone, dried, and then placed into the scintillation fluid. The data obtained from the spotting technique
VOL. 126, 1976
RIBOSOMAL PRECURSORS OF B. MEGATERIUM
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to both the classical precursors previously reported for other organisms (2, 10, 11, 27) and those obtained from DOC-treated protoplast lyRESULTS sates (Fig. 1B). The DOC-treated extracts gave sedimentation profiles (Fig. 2B) that were simiAnalysis of protoplast lysates. The intention of this study was to characterize the nature of lar to those previously reported, as well as the ribosomal subunit precursors in bacterial cells DOC-treated protoplast lysate (Fig. 1B). Howthat could be gently lysed. It was shown previ- ever, subtle differences may be seen if the unously that the use of a sucrose gradient formed treated whole-cell extract profile is compared to over a sucrose cushion made it possible to re- the DOC-treated whole-cell extract profile (Fig. from DOCsolve ribosomal subunits and, at the same time, 2). The pulse-labeled precursors observe faster-sedimenting material that would treated extracts have slightly lower sedimentaotherwise form a pellet (21). A modification of tion values than those from the untreated exthis technique was employed to analyze proto- tract. In addition, the 50S precursor stages plast lysates from pulse-labeled cells of B. meg- seem to be more discrete from the DOC-treated Furtheraterium. The sedimentation profiles of un- extract than the untreated extract. the DOCmature subunit from more, the 30s treated protoplast lysates compared with those lysates treated with DOC are illustrated in Fig. treated extract sedimented slightly slower than 1. Precursor particles (pulse label) from the lysates not treated with DOC cosedimented with mature 50S and 30S subunits. The usual "classical" sedimentation profiles observed in 3 3 studies with Escherichia coli (2, 10, 11) and Bacillus licheniformis (26, 27) were observed only when the lysate had been treated with 2 2 DOC (Fig. 1B). A considerable amount of both precursor and steady-state material from untreated protoplast lysates sedimented onto the 1 80% sucrose cushion (Fig. 1A); this was absent a-1 afrom the profile of DOC-treated lysates. FracU 2 tions containing material on the cushion from 0 untreated protoplast lysates (Fig. 1A) were iso0 lated from the sucrose gradients, pooled, and UI subsequently treated with DOC for examina3 I tion by sedimentation analysis. All of the steady-state and pulse-labeled material was distributed into the 50S and 30S regions of a 2 sucrose gradient profile identical to that observed in Fig. 1B (data not shown). Also, fractions containing the 50S RNP particles (Fig. 1 1A) were isolated from the sucrose gradients, individually treated with DOC, and then examined by sedimentation analysis. The RNP par10 20 30 ticles that were observed sedimented in the FRACTION NO. same positions as the pulse-labeled RNP particles from the DOC-treated protoplast lysates FIG. 1. Sucrose gradient sedimentation of unseen in Fig. 1B (data not shown). treated protoplast lysates (A) and treated with DOC Effect of cell breakage and detergent treat- (B). Sedimentation was from right to left. The protoment on the nature of RNP particles. The plast lysate was layered onto a 5 to 20% sucrose difference observed in the RNP particles after gradient in TM4 buffer formed over a 1.5-ml cushion DOC treatment of protoplast lysates led to fur- of 80% sucrose in TM4 (the arrow denotes the interbetween the cushion and the 20% sucrose). They ther studies on the effects of cell breakage tech- face were placed in 17-ml buckets of a Beckman SW27 niques and detergent. Cell extracts were pre- rotor and centrifuged at 25,000 rpm for 10.7 h at 5 C. pared with a French pressure cell. The prepara- The fractions were collected dropwise from the bottions were subsequently examined by sucrose tom of the gradient. Radioactivity of each fraction gradient analysis with and without prior DOC was determined by withdrawing 20-,l samples. treatment (Fig. 2). These ribosomal precursors Symbols: 0, ['4C]uracil (steady-state label); 0, from untreated extracts (Fig. 2A) were similar [3H]uridine (pulse label). were not significantly different from those obtained using co-precipitation with bovine serum albumin.
.
1152
BODY AND BROWNSTEIN
21 0-%0d
B
O U
J. BACTERIOL.
2H
50S
30S
C)~ ~ ~ ~ ~ 0 0
3
6I
2
4
10
20
FRACTION
30 NO.
FIG. 2. Sucrose gradient sedimentation of untreated whole-cell extracts (A) and DOC-treated whole-cell extracts (B). Sedimentation was from right to left. Extracts were prepared from whole cells broken by passage through a French press. Samples were layered onto 5 to 20% sucrose gradients in TM4 buffer formed over a 1 .5-ml cushion of80% sucrose in TM4 buffer (the arrow denotes the interface between the cushion and the 20% sucrose). They were placed in 1 7-ml buckets of a Beckman SW27 rotor and centrifuged at 24,000 rpm for 10.7 h at 5 C. The fractions were collected dropwise from the bottom of the gradient, precipitated, and counted. Symbols: 0, ['4C]uracil (steady-state label); 0, [3H]uridine (pulse label).
the mature 30S subunit from the untreated extract. This experiment was repeated two more times with the same result. Effect of lysozyme on precursor particles. Patterson et al. (20) demonstrated that lysozyme could mediate the binding of ribosomes to cell fragments. Furthermore, Dimmitt and Sypherd (4) showed that in the presence of lysozyme 43S RNP particles bind this protein and subsequently sediment at the 505 subunit position. Their study also showed that the binding of lysozyme could be prevented or reversed by the presence of 50 mM KCl (4). Therefore, a portion of the whole-cell extract (Fig. 2A) was incubated with lysozyme at 200 ,ug/ml and subsequently analyzed by sucrose gradient sedimentation to determine whether lysozyme
binding might have occurred in the experiments reported here. The sedimentation profile of the lysozyme-treated extract (Fig. 3A) was not significantly different from that of the untreated control profile (Fig. 3B). Polyacrylamide gel analysis of RNA from precursor particles. To demonstrate that the 50S and 30S pulse-labeled RNP particles observed in gradients of protoplast lysates (Fig. 1A) were precursor particles, the rRNA that they contained was analyzed by polyacrylamide gel electrophoresis. The 50S and 305 subunit peak fractions were pooled (fractions 13 through 16 and 22 through 24, respectively, of Fig. 1A) and subsequently treated with SDS prior to layering them on the gels for electrophoresis. A portion of the original protoplast lysate was treated with SDS and subsequently subjected to polyacrylamide gel electrophoresis to serve as a control. The electrophoretic profile of the treated protoplast lysate revealed that the pulse label was distributed predominantly in the regions of the gel normally associated with precursor 23S and 16S rRNA (Fig. 4A). When the aforementioned 505 or 30S ppak
ia-
0s
U 0
u 0
Iv -r
U..
10
20
3(
FRACTION NO. FIG. 3. Sucrose gradient sedimentation of wholecell extracts incubated with lysozyme at 200 jg/ml (A) and the control (B). After incubation, samples were layered onto 16.5-ml 5 to 20% sucrose gradients in TM4 buffer. The gradients were placed in 17-ml buckets of a Beckman SW27 rotor and centrifuged for 10.7 h at 5 C. The fractions were collected from the bottom of the gradient, precipitated, and counted. Symbols: 0, ["4C]uracil (steady-state label); 0, [3H]uridine (pulse label).
RIBOSOMAL PRECURSORS OF B. MEGATERIUM
VOL. 126, 1976
T
ELL 6
50S PE,AK
,> 2
a_
° °
U
04
3
^ fPEAK ~~~~~~~30S
C 2
6
3 45
55
85
95
105
Gel Fraction No.
FIG. 4. Polyacrylamide gel electropihioresis of RNAs from whole-cell extract (A), 50S wature subunits and RNP particles (B), and 30S mLature subunits and RNP particles (C). Electroph oresis was from left to right. The RNA from the whtole-cell extract was solubilized by the addition of ISDS (0.2% final concentration). The RNA in the stucrose gradient fractions containing either the 50S nnature suband units and RNP particles or 30S mature su RNP particles (see Fig. 1A) was also sol ubilized by SDS. Glycerol was then added, and the sa layered on 2.8% polyacrylamide gels. Elec trophoresis was for 5.5 h at 10 mA/gel at 22 C. After electrophoresis the gels were frozen, sliced into O.'5-mm fractions, and counted. Symbols: *, ['4C]ura cil (steadystate label); 0, [3H]uridine (pulse label).
cbunits
fractions were treated with SDS and subjected to electrophoresis, the pulse label wass observed in both the precursor and mature pc)sitions of the respective 23S or 16S rRNA regi ons of the gel (Fig. 4B and C).
DISCUSSION It is apparent from the experiment: s reported here that the precursor ribosomal sulbunit profile is dependent on the procedure us,ed for cell extract preparation. The method of c ell breakage or the use of DOC can influence t,he nature or structure of the subunit precursor s from B. megaterium. The analysis of extracts3 from untreated protoplast lysates (Fig. 1) indlicated the presence of 50S and 30S precursor piarticles in B. megaterium similar to those ob)served in extracts of E. coli by Lindahl (9) arid Mangiarotti et al. (13). However, DOC treaitment al. ..--Cl tered the sedimentation values of the B.rnega-
1153
terium 50S and 30S RNP particles such that their new locations in the gradient were those positions usually associated with the major precursor ribosomal subunits reported for other organisms (2, 10, 11, 25). Some pulse-labeled material from the untreated protoplast lysate was found at the bottom of the sucrose gradient on the 80% sucrose cushion (Fig. 1A). When this fraction was DOC treated and then analyzed by sedimentation, the pulse label was observed in 50S and 30S RNP particles. Since the steady-state label seen in mature 50S and 30S subunits was also found on the cushion, it is possible that the 50S and 30S RNP particles were part of some type of aggregate. Another possibility is that material found on the cushion is the remnant of the polysome fraction and that the 50S and 30S RNP particles ofB. megaterium are associated with polysomes in vivo. Polyacrylamide gel analysis of the RNA from the isolated 50S and 30S RNP particles confirmed their precursor nature (Fig. 4). These particles contained both precursor and mature forms of 23S or 16S RNA, in contrast to the rRNA in the crude protoplast lysate which was almost completely in the precursor form. The 50S and 30S particles should have contained only precursor rRNA, but it is possible that some of it was converted to the mature form by an endogenous ribonuclease(s) during sedimentation and subsequent isolation. Hayes and Hayes (6) demonstrated the presence of partially processed rRNA in ribosomal precursor particles of E. coli. It is not known whether conversion of the precursor rRNA in B. megaterium involved a specific or nonspecific cleavage of the rRNA. The effects of the method of cell disruption and DOC treatment on ribosomal precursors were examined further in whole-cell extracts, which were prepared using a French pressure cell (Fig. 2). In these extracts, the particles that moved slower than 50S and 30S RNP were again observed. Although the sedimentation profiles of the untreated and DOC-treated extracts were similar, a comparison of these profiles revealed that the DOC-treated precursors were again observed at positions of slightly lower sedimentation values. Therefore, the detergent consistently has an effect on all precursor-type particles. The presence of the 50S and 30S precursors observed in the protoplast lysate could have resulted from the binding of lysozyme (4, 20). However, the sedimentation profile of the lysozyme-treated extract was not significantly different from that of the untreated control (Fig. 3), indicating that lysozyme was not responsible.
1154
BODY AND BROWNSTEIN
J. BACTERIOL.
All previous studies that have reported ACKNOWLEDGMENTS slower-sedimenting precursors have always We are indebted to Joseph Wachsman for supplying us employed vigorous cell breakage techniques or with B. megaterium KM. We wish to thank Paul Sypherd the combination of gentle lysis and detergent and David Schlessinger for some very helpful and interestdiscussions concerning the evaluation of the data. treatment. The experiments reported here have ingThis investigation was supported by a General Research shown that it is possible to generate "classical" Support Grant to B. H. Brownatein from the College of precursors to subunits by several methods. Medicine, University of Kentucky. These results lead to a serious question conLITERATURE CITED cerning the true in vivo nature of ribosomal precursors. The 5as- and 30S-type precursors 1. Bishop, D. H. L., J. R. Claybrook, and S. Spiegelman. 1967. Electrophoretic separation of viral nucleic acids probably represent a more compact configuraon polyacrylamide gels. J. Mol. Biol. 26:373-387. tion of the classical precursor particles. Per- 2. Britten, R. J., B. J. McCarthy, and R. B. Roberts. 1962. haps the conformation of these particles is easThe synthesis of ribosomes in E. coli. IV. The synthesis of ribosomal protein and the assembly of riboily disrupted during cell extract preparation J. 2:83-93. (by either DOC treatment or the high shear 3. somes.R.Biophys. Bryant, E., and P. S. Sypherd. 1974. Genetic analyforces involved in mechanical breakage of sis of cold-sensitive ribosome maturation mutants of cells). DOC could alter the conformation of the Ewcherichia coli. J. Bacteriol. 117:1082-1092. precursors by causing them to unfold, removing 4. Dimmitt, K., and P. S. Sypherd. 1973. Association of lysozyme with ribosomes and ribosomal precursor ribosomal proteins, or a combination of both. particles. J. Bacteriol. 116:1059-1061. This is consistent with the observations of 5. Guthrie, C. H., H. Nashimoto, and M. Nomura. 1969. Hayes and Hayes (6) and Lindahl (9), who atStructure and function of E. coli ribosomes. VIII. Cold-sensitive mutants defective in ribosome assemtributed differences in the reported sedimentaNatl. Acad. Sci. U.S.A. 63:384-391. tion values of the precursors to their instabil- 6. bly. Proc. Hayes, F., and D. H. Hayes. 1971. Biosynthesis of riboity. Another possibility is that the 5SS and 30S somes in E. coli. I. Properties of ribosomal precursor precursors are the actual precursors but have particles and their RNA components. Biochimie 53:369-382. small fragments of membrane associated with L. J., and B. L. Browstein. 1969. Charthem. Vigorous lysis or DOC treatment might 7. Lewandowski, acterization of a 43S ribo-nucleoprotein component of disrupt such complexes, whereas gentle lysis a mutant ofEscherichia coli. J. Mol. Biol. 41:277-290. without detergent could leave them intact. 8. Lindahl, L. 1973. Two new ribosomal precursor particles in E. coli. Nature (London) New Biol. 243:170The results of this study are compatible with 172. two recent independent studies on the in vitro 9. Lindahl, L. 1975. Intermediate and time kinetics of the reconstitution of ribosomal subunits from prein vivo assembly of Escherichia coli ribosomes. J. cursor rRNA species. Mangiarotti et al. (12) Mol. Biol. 92:15-37. reported the reconstitution of a 30S subunit 10. McCarthy, B. J., R. J. Britten, and R. B. Roberts. 1962. The synthesis ofribosomes inE. coli. JIl. Synthesis of without the formation of an intermediate partiribosomal RNA. Biophys. J. 2:57-82. cle when they used precursor 16S rRNA and 11. Mangiarotti, G., D. Apirion, D. Schlewinger, and L. proteins from nascent ribosomes. In the second Silengo. 1968. Biosynthetic precursors of 30S and 505 ribosomal particles in Escherichia coli. Biochemistry study, Nikolaev et al. (16) reconstituted a 53S 7:456-472. particle by incubating the large precursor 30S 12. Mangiarotti, G., E. Turco, C. Perlo, and F. Altmda. rRNA with the complete mixture of 50S and 1975. Role ofprecursor 16S RNA in assembly of E. coli 30S proteins. If the 53S reconstituted particle is 30S ribosomes. Nature (London) 263:569-570. then incubated with ribonuclease HI, both 50S 13. Mangiarottl, G., E. Turco, A. Ponzetto, and F. Altruda. 1974. Precursor 16S RNA in active 30S riboand 30S precursor particles are produced. All of somes. Nature (London) 247:147-148. these precursor particles are thus formed with- 14. Nashimoto, H., W. Held, E. Kaldschmidt, and M. Noout any observed slower-moving intermediate. mura 1971. Structure and function of bacterial ribosomes. XII. Accumulation of 21S particles by some It is possible that the regions of the sucrose cold-sensitive mutants of Escherichia coli. J. Mol. gradients that contain the 5OS and 30S precurBiol. 62:121-138. sor particles reported here and by Lindahl (9) 15. Nierhaus, K., K. Bordasch, and H. Hon'an. 1973. Riboinclude not only the earliest stages of assembly somal proteins. XLII. In vivo assembly of Escherichia coli ribosomal proteins. J. Mol. Biol. 74:587but later stages as well. All of them can be 597. indistinguishable by sedimentation (i.e., they 16. Nikolsev, N., D. Glazier, and D. Schlesinger. 1975. still sediment at 50S and 30S) if the cell exCleavage by ribonuclease III of the complex of 30S tracts are produced by gentle means. The heterpre-ribosomal RNA and ribosomal proteins of Escherichaa coli. J. Mol. Biol. 94:3014304. ogeneity of the 50S and 30S precursor partiN., and D. Schlesinger. 1974. Binding of cles-and their intrinsic lack of compactness 17. Nikolnev, ribosomal proteins to 308 pre-ribosomal ribonucleic compared to mature subunits -would then be acid ofEscherichia coli. Biochemistry 13:4272-4278. revealed when bacterial cells are disrupted by 18. Nomura, M., and V. A. Erdmann. 1970. Reconstitution ofthe 505 ribosomal subunits from dissociated molecharsh means or through the use of a detergent.
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21.
22. 23.
RIBOSOMAL PRECURSORS OF B. MEGATERIUM
ular components. Nature (London) 228:744-748. Osawa, S., E. Otaka, T. Itoh, and T. Fukui. 1969. Biosynthesis of 50S ribosomal subunit in Escherichia coli. J. Mol. Biol. 40:321-351. Patterson, D., M. Weinstein, R. Nixon, and D. Gillepsie. 1970. Interaction of ribosomes and the cell envelope of Escherichia coli mediated by lysozyme. J. Bacteriol. 101:584-591. Roth, G. S., and L. Daneo-Moore. 1971. Intracellular location of ribosome biosynthesis in osmotically fragile forms of Streptococcus faecalis. Biochim. Biophys. Acta 240:575-587. Sypherd, P. S., R. E. Bryant, K. Dimmitt, and P. Fujisawa. 1974. Genetic control of ribosome assembly. J. Supramol. Struct. 2:166-177. Tai, P.-C., D. P. Kessler, and J. Ingraham. 1969. Coldsensitive mutations in Salmonella typhimurium which affect ribosomes synthesis. J. Bacteriol.
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24. Traub, P., and M. Nomura. 1969. Structure and function of Escherichia coli ribosomes. VI. Mechanism of assembly of 30S ribosomes studies in vitro. J. Mol. Biol. 40:391-413. 25. Tremblay, G. Y., M. J. Daniels, and M. Schaecter. 1969. Isolation of a cell membrane-DNA-nascent RNA complex from bacteria. J. Mol. Biol. 40:65-76. 26. Van-Dijk-Salkinoja, M. S., and R. J. Planta. 1970. Formation and life cycle of ribosomal subunits in Bacillus licheniformis. Arch. Biochem. Biophys. 141:477488. 27. Van-Dijk-Salkinoja, M. S., T. J. Stoof, and R. J. Planta. 1970. The distribution of polysomes, ribosomes, and ribosomal subunits in exponential-phase cells of Bacillus licheniformis. Eur. J. Biochem. 12:474-482. 28. Wachsman, J. T., and R. Storch. 1965. Propionateinduced lysis of protoplasts of Bacillus megaterium. J. Bacteriol. 80:600-606.
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Vol. 126, No. 3 Printed in U.S.A.
BACTERIOLOGY, June 1976, p. 1149-1155
Copyright © 1976 American Society for Microbiology
Ribosomal Precursor Particles of Bacillus megaterium BARBARA A. BODY AND BERNARD H. BROWNSTEIN* Department of Cell Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40506
Received for publication 1 March 1976
Pulse-labeled cells ofBacillus megaterium were converted to protoplasts, and lysates of the protoplasts were analyzed by sucrose gradient sedimentation. Precursor ribonucleoprotein (RNP) particles then appeared predominantly as 50S and 30S precursor ribosomal subunits. Polyacrylamide gel electrophoresis of the ribosomal ribonucleic acid from the 50S and 30S RNP particles confirmed their precursor nature since they were shown to contain precursor 23S and 16S ribosomal ribonucleic acid, respectively. Treatment of protoplast lysates with 0.5% deoxycholate prior to sedimentation analysis resulted in a markedly different radioactivity profile. The 50S RNP particles were no longer present, but 43S particles were observed in addition to increased amounts of pulse-labeled material sedimenting at 30S and slower. Extracts from cells broken in a French press showed a profile from sucrose gradient sedimentation similar to that of the deoxycholate-treated protoplast lysate. These data suggest that the nature ofthe precursor ribosomal particles appears to be a function of the method of cell disruption or detergent treatment of the cell extract preparation. The observed 509 and 30S RNP particles may be the major precursor ribosomal subunits in vivo; the slower-sedimenting species could result from some form of breakdown or change in the configuration of the 50S and 30S precursors. Britten et al. (2, 10) first demonstrated the existence of precursor ribosomal subunits. Further work by Mangiarotti et al. (11) identified three ribosomal subunit precursors, two for the 50S subunit and one for the 30S subunit. Other studies of various mutants that accumulate ribonucleoprotein particles (RNP) corresponding to the classical ribosomal precursor stages (4-7, 9-11) contributed to the hypothesis that these precursors may correspond to ratelimiting steps in the subunit assembly process. Ribosome assembly has also been examined by characterizing in vitro reconstitution of subunits (12, 18, 24). An intermediate particle was observed when mature 16S ribosomal ribonucleic acid (rRNA) and 30S proteins were used for reconstitution of 30S ribosomal subunits. However, this intermediate has a somewhat different protein composition than that of the in vivo 30s precursor (15). Conversion of this particle to a functional 30S subunit was shown to be temperature dependent (24). Also, Mangiarotti et al. (12) successfully used precursor 16S rRNA in the reconstitution of active 30S subunits and showed that it is not temperature dependent and has no intermediate like that observed with mature rRNA. Thus, the in vivo assembly process seems somewhat different than that inferred from in vitro reconstitution systems.
Recently, two new ribosomal precursors that cosediment with the mature 50S and 30S subunits were reported by Lindahl (8, 9). The slower-sedimenting ribosomal precursors were also observed, but it was concluded that the 50S and 30S cosedimenting particles were the major precursor species and that they were not observed in other studies because of their relative instability. Variations in observed sedimentation values of classical precursor particles have been reported (6, 19) which could also be due to instability. In this study of ribosomal precursors ofBacillus megaterium we have also demonstrated that the major precursor particles cosediment with the mature 50S and 30S subunits. Furthermore, the sedimentation of ribosomal precursors observed was, in fact, dependent on the method of cell disruption employed. Vigorous and mild cell breakage techniques, as well as detergent treatments, have a profound effect on the properties of the ribosomal precursor particles. MATERIALS AND METHODS Bacterium and growth of cultures. The bacterium used throughout this study was B. megaterium KM. Stock cultures of the organism were maintained on slants of stock culture medium (Difco Laboratories). In all experiments the bacteria were
1149
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grown in the minimal medium of Wachsman and Storch (28) modified to contain 0.5% glucose and 20% (wt/vol) sucrose and supplemented with 40 ,ug of Larginine per ml. All cultures were grown at 30 C in a gyratory shaker that rotated at 250 rpm. They exhibited a 60-min generation time. GrQwth was monitored with a Klett-Summerson colorimeter using a no. 54 filter (540 nm); one Klett unit equaled 9 x 105 cells per ml. Isotopic labeling of cells. Flasks containing fresh medium were inoculated with a 1:100 dilution from an overnight culture. After one doubling, cells were steady-state labeled by the addition of 0.2 ,uCi of [2'4C]uracil (specific activity, 56 mCi/mmol) per ml to the culture. After four more generations, cells were pulse labeled for 2.5 min by the addition of 2 ,uCi of [5,6-3H]uridine (specific activity, 26 Ci/mmol) per ml. At the end of the 2.5-min pulse period, sodium azide and chloramphenicol were added to final concentrations of 1 mM and 100 yg/ml, respectively. Cells prepared in this manner were either converted to protoplasts or subjected to the treatments described below. Preparation of protoplasts and protoplast lysates. Protoplast formation was accomplished by the procedure of Tremblay et al. (25), with the following modifications. The sodium azide- and chloramphenicol-treated cells were immediately poured into a prewarmed Erlenmeyer flask that contained lysozyme (EC 3.2.1.17) such that the final concentration would be 200 ,ug/ml. The flask was then swirled in a 37 C water bath for 30 s. The suspension was then poured over a 4/5 volume of crushed, frozen TM2KS buffer [10 mM tris(hydroxymethyl)aminomethanehydrochloride, pH 7.2; 10 mM magnesium acetate; 100 mM KCl; 20% (wt/vol) sucrose]. All subsequent steps were carried out at 0 to 4 C. Protoplasts were harvested by centrifugation at 6,000 x g for 7 min in a Sorvall RC2-B centrifuge. The supernatant fluid was discarded. The pellet of protoplasts was suspended and lysed in cold TM4K buffer [10 mM
tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.2; 0.1 mM magnesium acetate; 50 mM KClI containing deoxyribonuclease (EC 3.1.4.5) at 10 ,ug/ ml. In those experiments in which an alternate technique was used to break protoplasts, the pellet was suspended in TM4K buffer containing 10% (wt/ vol) sucrose and immediately broken by the alternative technique. Preparation of whole-cell extracts by using a French pressure cell. Cells not converted to protoplasts were treated in a manner identical to that described above, except that lysozyme was omitted. Resuspended cells or protoplasts were broken by passage through a chilled French pressure cell at 10,000 lb/in2. The broken cell preparation was collected in a chilled tube containing deoxyribonuclease at 10 ,ig/ml. After protoplasts were broken by this procedure, the preparation was subsequently diluted by adding 3 volumes of TM4K buffer. All lysates and extracts were either used immediately or frozen and stored at -70 C. The data obtained from stored extracts did not differ significantly from those obtained from extracts used immediately. Detergent and lysozyme treatment of lysates.
J. BACTERIOL.
The portions of protoplast lysates or cell extracts that were to be detergent or lysozyme treated were incubated with sodium deoxycholate (DOC) at 0.5% or lysozyme at 200 ,ug/ml for 15 min at 0 C prior to sedimentation analysis. Sucrose gradient sedimentation analysis. Ribosomal subunits and precursor particles were separated by centrifugation of the protoplast lysate or cell extracts through linear 5 to 20% (wt/vol) sucrose-TM4 (TM4K without KCl) gradients. The gradients were formed over a 1.5-ml 80% (wt/vol) sucrose-TM4 cushion used to catch material that might otherwise form a pellet (21). A Beckman SW27 rotor with 17-ml tubes was used for all sedimentation analysis. After 10.7 h of centrifugation at 4 C, the tubes were punctured at the bottom with a needle in a drop-collecting apparatus to collect fractions. Usually 40 fractions were obtained per gradient. RNA analysis of lysates, extracts, and particles. The sucrose gradient fractions containing either the 50S or 30S subunits or precursor particles were individually pooled and subsequently precipitated by raising the magnesium ion concentration to 20 mM and adding 0.7 volume of cold ethanol. After 30 min at -20 C, the precipitates were collected by centrifugation at 12,000 x g for 30 min in the cold. The pellets were then suspended in ASES buffer (10 mM sodium acetate, pH 5.2; 100 mM sodium chloride; 1 mM disodium ethylenediaminetetraacetate [EDTA]; 0.2% sodium dodecyl sulfate [SDS]). When appropriate, the crude cell extracts and protoplast lysates were prepared for RNA analysis by the addition of 0.2% SDS (final concentration). Polyacrylamide gel electrophoresis. The method of Bishop et al. (1) was used for RNA analysis. The samples were layered on cylindrical gels (9 by 90 mm) and developed by regulating the current at 10 mA/gel in E buffer (1). The developed gels were frozen at - 40 C on aluminum trays prior to preparation for radioactive analysis. A Mickel gel slicer was used to obtain 0.5-mm slices, which were subsequently dried on GF/A paper (Reeve-Angel). The paper was then placed in scintillation vials and the radioactivity was determined as described below. Determination of radioactivity. The radioactivity of gradient fractions was measured by one of two methods. In the first method, 200 ,ug ofbovine serum albumin (Miles Laboratories; purified fraction V) was added to each fraction and then precipitated with cold 5% trichloroacetic acid. The precipitates were collected on GF/A filters and washed three times with cold 5% trichloroacetic acid. They were then dried, put into glass scintillation vials containing toluene-Omnifluor scintillation fluid (New England Nuclear Corp.), and counted in a Packard TriCarb liquid scintillation spectrometer, model 3375. The second method was utilized only when fractions were also to be used for RNA analysis. In this case, a 20-,Al sample from each fraction was spotted on a GF/C filter and placed into 5% trichloroacetic acid. The filters were rinsed with three changes of 5% trichloroacetic acid followed by three rinses with acetone, dried, and then placed into the scintillation fluid. The data obtained from the spotting technique
VOL. 126, 1976
RIBOSOMAL PRECURSORS OF B. MEGATERIUM
1151
to both the classical precursors previously reported for other organisms (2, 10, 11, 27) and those obtained from DOC-treated protoplast lyRESULTS sates (Fig. 1B). The DOC-treated extracts gave sedimentation profiles (Fig. 2B) that were simiAnalysis of protoplast lysates. The intention of this study was to characterize the nature of lar to those previously reported, as well as the ribosomal subunit precursors in bacterial cells DOC-treated protoplast lysate (Fig. 1B). Howthat could be gently lysed. It was shown previ- ever, subtle differences may be seen if the unously that the use of a sucrose gradient formed treated whole-cell extract profile is compared to over a sucrose cushion made it possible to re- the DOC-treated whole-cell extract profile (Fig. from DOCsolve ribosomal subunits and, at the same time, 2). The pulse-labeled precursors observe faster-sedimenting material that would treated extracts have slightly lower sedimentaotherwise form a pellet (21). A modification of tion values than those from the untreated exthis technique was employed to analyze proto- tract. In addition, the 50S precursor stages plast lysates from pulse-labeled cells of B. meg- seem to be more discrete from the DOC-treated Furtheraterium. The sedimentation profiles of un- extract than the untreated extract. the DOCmature subunit from more, the 30s treated protoplast lysates compared with those lysates treated with DOC are illustrated in Fig. treated extract sedimented slightly slower than 1. Precursor particles (pulse label) from the lysates not treated with DOC cosedimented with mature 50S and 30S subunits. The usual "classical" sedimentation profiles observed in 3 3 studies with Escherichia coli (2, 10, 11) and Bacillus licheniformis (26, 27) were observed only when the lysate had been treated with 2 2 DOC (Fig. 1B). A considerable amount of both precursor and steady-state material from untreated protoplast lysates sedimented onto the 1 80% sucrose cushion (Fig. 1A); this was absent a-1 afrom the profile of DOC-treated lysates. FracU 2 tions containing material on the cushion from 0 untreated protoplast lysates (Fig. 1A) were iso0 lated from the sucrose gradients, pooled, and UI subsequently treated with DOC for examina3 I tion by sedimentation analysis. All of the steady-state and pulse-labeled material was distributed into the 50S and 30S regions of a 2 sucrose gradient profile identical to that observed in Fig. 1B (data not shown). Also, fractions containing the 50S RNP particles (Fig. 1 1A) were isolated from the sucrose gradients, individually treated with DOC, and then examined by sedimentation analysis. The RNP par10 20 30 ticles that were observed sedimented in the FRACTION NO. same positions as the pulse-labeled RNP particles from the DOC-treated protoplast lysates FIG. 1. Sucrose gradient sedimentation of unseen in Fig. 1B (data not shown). treated protoplast lysates (A) and treated with DOC Effect of cell breakage and detergent treat- (B). Sedimentation was from right to left. The protoment on the nature of RNP particles. The plast lysate was layered onto a 5 to 20% sucrose difference observed in the RNP particles after gradient in TM4 buffer formed over a 1.5-ml cushion DOC treatment of protoplast lysates led to fur- of 80% sucrose in TM4 (the arrow denotes the interbetween the cushion and the 20% sucrose). They ther studies on the effects of cell breakage tech- face were placed in 17-ml buckets of a Beckman SW27 niques and detergent. Cell extracts were pre- rotor and centrifuged at 25,000 rpm for 10.7 h at 5 C. pared with a French pressure cell. The prepara- The fractions were collected dropwise from the bottions were subsequently examined by sucrose tom of the gradient. Radioactivity of each fraction gradient analysis with and without prior DOC was determined by withdrawing 20-,l samples. treatment (Fig. 2). These ribosomal precursors Symbols: 0, ['4C]uracil (steady-state label); 0, from untreated extracts (Fig. 2A) were similar [3H]uridine (pulse label). were not significantly different from those obtained using co-precipitation with bovine serum albumin.
.
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BODY AND BROWNSTEIN
21 0-%0d
B
O U
J. BACTERIOL.
2H
50S
30S
C)~ ~ ~ ~ ~ 0 0
3
6I
2
4
10
20
FRACTION
30 NO.
FIG. 2. Sucrose gradient sedimentation of untreated whole-cell extracts (A) and DOC-treated whole-cell extracts (B). Sedimentation was from right to left. Extracts were prepared from whole cells broken by passage through a French press. Samples were layered onto 5 to 20% sucrose gradients in TM4 buffer formed over a 1 .5-ml cushion of80% sucrose in TM4 buffer (the arrow denotes the interface between the cushion and the 20% sucrose). They were placed in 1 7-ml buckets of a Beckman SW27 rotor and centrifuged at 24,000 rpm for 10.7 h at 5 C. The fractions were collected dropwise from the bottom of the gradient, precipitated, and counted. Symbols: 0, ['4C]uracil (steady-state label); 0, [3H]uridine (pulse label).
the mature 30S subunit from the untreated extract. This experiment was repeated two more times with the same result. Effect of lysozyme on precursor particles. Patterson et al. (20) demonstrated that lysozyme could mediate the binding of ribosomes to cell fragments. Furthermore, Dimmitt and Sypherd (4) showed that in the presence of lysozyme 43S RNP particles bind this protein and subsequently sediment at the 505 subunit position. Their study also showed that the binding of lysozyme could be prevented or reversed by the presence of 50 mM KCl (4). Therefore, a portion of the whole-cell extract (Fig. 2A) was incubated with lysozyme at 200 ,ug/ml and subsequently analyzed by sucrose gradient sedimentation to determine whether lysozyme
binding might have occurred in the experiments reported here. The sedimentation profile of the lysozyme-treated extract (Fig. 3A) was not significantly different from that of the untreated control profile (Fig. 3B). Polyacrylamide gel analysis of RNA from precursor particles. To demonstrate that the 50S and 30S pulse-labeled RNP particles observed in gradients of protoplast lysates (Fig. 1A) were precursor particles, the rRNA that they contained was analyzed by polyacrylamide gel electrophoresis. The 50S and 305 subunit peak fractions were pooled (fractions 13 through 16 and 22 through 24, respectively, of Fig. 1A) and subsequently treated with SDS prior to layering them on the gels for electrophoresis. A portion of the original protoplast lysate was treated with SDS and subsequently subjected to polyacrylamide gel electrophoresis to serve as a control. The electrophoretic profile of the treated protoplast lysate revealed that the pulse label was distributed predominantly in the regions of the gel normally associated with precursor 23S and 16S rRNA (Fig. 4A). When the aforementioned 505 or 30S ppak
ia-
0s
U 0
u 0
Iv -r
U..
10
20
3(
FRACTION NO. FIG. 3. Sucrose gradient sedimentation of wholecell extracts incubated with lysozyme at 200 jg/ml (A) and the control (B). After incubation, samples were layered onto 16.5-ml 5 to 20% sucrose gradients in TM4 buffer. The gradients were placed in 17-ml buckets of a Beckman SW27 rotor and centrifuged for 10.7 h at 5 C. The fractions were collected from the bottom of the gradient, precipitated, and counted. Symbols: 0, ["4C]uracil (steady-state label); 0, [3H]uridine (pulse label).
RIBOSOMAL PRECURSORS OF B. MEGATERIUM
VOL. 126, 1976
T
ELL 6
50S PE,AK
,> 2
a_
° °
U
04
3
^ fPEAK ~~~~~~~30S
C 2
6
3 45
55
85
95
105
Gel Fraction No.
FIG. 4. Polyacrylamide gel electropihioresis of RNAs from whole-cell extract (A), 50S wature subunits and RNP particles (B), and 30S mLature subunits and RNP particles (C). Electroph oresis was from left to right. The RNA from the whtole-cell extract was solubilized by the addition of ISDS (0.2% final concentration). The RNA in the stucrose gradient fractions containing either the 50S nnature suband units and RNP particles or 30S mature su RNP particles (see Fig. 1A) was also sol ubilized by SDS. Glycerol was then added, and the sa layered on 2.8% polyacrylamide gels. Elec trophoresis was for 5.5 h at 10 mA/gel at 22 C. After electrophoresis the gels were frozen, sliced into O.'5-mm fractions, and counted. Symbols: *, ['4C]ura cil (steadystate label); 0, [3H]uridine (pulse label).
cbunits
fractions were treated with SDS and subjected to electrophoresis, the pulse label wass observed in both the precursor and mature pc)sitions of the respective 23S or 16S rRNA regi ons of the gel (Fig. 4B and C).
DISCUSSION It is apparent from the experiment: s reported here that the precursor ribosomal sulbunit profile is dependent on the procedure us,ed for cell extract preparation. The method of c ell breakage or the use of DOC can influence t,he nature or structure of the subunit precursor s from B. megaterium. The analysis of extracts3 from untreated protoplast lysates (Fig. 1) indlicated the presence of 50S and 30S precursor piarticles in B. megaterium similar to those ob)served in extracts of E. coli by Lindahl (9) arid Mangiarotti et al. (13). However, DOC treaitment al. ..--Cl tered the sedimentation values of the B.rnega-
1153
terium 50S and 30S RNP particles such that their new locations in the gradient were those positions usually associated with the major precursor ribosomal subunits reported for other organisms (2, 10, 11, 25). Some pulse-labeled material from the untreated protoplast lysate was found at the bottom of the sucrose gradient on the 80% sucrose cushion (Fig. 1A). When this fraction was DOC treated and then analyzed by sedimentation, the pulse label was observed in 50S and 30S RNP particles. Since the steady-state label seen in mature 50S and 30S subunits was also found on the cushion, it is possible that the 50S and 30S RNP particles were part of some type of aggregate. Another possibility is that material found on the cushion is the remnant of the polysome fraction and that the 50S and 30S RNP particles ofB. megaterium are associated with polysomes in vivo. Polyacrylamide gel analysis of the RNA from the isolated 50S and 30S RNP particles confirmed their precursor nature (Fig. 4). These particles contained both precursor and mature forms of 23S or 16S RNA, in contrast to the rRNA in the crude protoplast lysate which was almost completely in the precursor form. The 50S and 30S particles should have contained only precursor rRNA, but it is possible that some of it was converted to the mature form by an endogenous ribonuclease(s) during sedimentation and subsequent isolation. Hayes and Hayes (6) demonstrated the presence of partially processed rRNA in ribosomal precursor particles of E. coli. It is not known whether conversion of the precursor rRNA in B. megaterium involved a specific or nonspecific cleavage of the rRNA. The effects of the method of cell disruption and DOC treatment on ribosomal precursors were examined further in whole-cell extracts, which were prepared using a French pressure cell (Fig. 2). In these extracts, the particles that moved slower than 50S and 30S RNP were again observed. Although the sedimentation profiles of the untreated and DOC-treated extracts were similar, a comparison of these profiles revealed that the DOC-treated precursors were again observed at positions of slightly lower sedimentation values. Therefore, the detergent consistently has an effect on all precursor-type particles. The presence of the 50S and 30S precursors observed in the protoplast lysate could have resulted from the binding of lysozyme (4, 20). However, the sedimentation profile of the lysozyme-treated extract was not significantly different from that of the untreated control (Fig. 3), indicating that lysozyme was not responsible.
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J. BACTERIOL.
All previous studies that have reported ACKNOWLEDGMENTS slower-sedimenting precursors have always We are indebted to Joseph Wachsman for supplying us employed vigorous cell breakage techniques or with B. megaterium KM. We wish to thank Paul Sypherd the combination of gentle lysis and detergent and David Schlessinger for some very helpful and interestdiscussions concerning the evaluation of the data. treatment. The experiments reported here have ingThis investigation was supported by a General Research shown that it is possible to generate "classical" Support Grant to B. H. Brownatein from the College of precursors to subunits by several methods. Medicine, University of Kentucky. These results lead to a serious question conLITERATURE CITED cerning the true in vivo nature of ribosomal precursors. The 5as- and 30S-type precursors 1. Bishop, D. H. L., J. R. Claybrook, and S. Spiegelman. 1967. Electrophoretic separation of viral nucleic acids probably represent a more compact configuraon polyacrylamide gels. J. Mol. Biol. 26:373-387. tion of the classical precursor particles. Per- 2. Britten, R. J., B. J. McCarthy, and R. B. Roberts. 1962. haps the conformation of these particles is easThe synthesis of ribosomes in E. coli. IV. The synthesis of ribosomal protein and the assembly of riboily disrupted during cell extract preparation J. 2:83-93. (by either DOC treatment or the high shear 3. somes.R.Biophys. Bryant, E., and P. S. Sypherd. 1974. Genetic analyforces involved in mechanical breakage of sis of cold-sensitive ribosome maturation mutants of cells). DOC could alter the conformation of the Ewcherichia coli. J. Bacteriol. 117:1082-1092. precursors by causing them to unfold, removing 4. Dimmitt, K., and P. S. Sypherd. 1973. Association of lysozyme with ribosomes and ribosomal precursor ribosomal proteins, or a combination of both. particles. J. Bacteriol. 116:1059-1061. This is consistent with the observations of 5. Guthrie, C. H., H. Nashimoto, and M. Nomura. 1969. Hayes and Hayes (6) and Lindahl (9), who atStructure and function of E. coli ribosomes. VIII. Cold-sensitive mutants defective in ribosome assemtributed differences in the reported sedimentaNatl. Acad. Sci. U.S.A. 63:384-391. tion values of the precursors to their instabil- 6. bly. Proc. Hayes, F., and D. H. Hayes. 1971. Biosynthesis of riboity. Another possibility is that the 5SS and 30S somes in E. coli. I. Properties of ribosomal precursor precursors are the actual precursors but have particles and their RNA components. Biochimie 53:369-382. small fragments of membrane associated with L. J., and B. L. Browstein. 1969. Charthem. Vigorous lysis or DOC treatment might 7. Lewandowski, acterization of a 43S ribo-nucleoprotein component of disrupt such complexes, whereas gentle lysis a mutant ofEscherichia coli. J. Mol. Biol. 41:277-290. without detergent could leave them intact. 8. Lindahl, L. 1973. Two new ribosomal precursor particles in E. coli. Nature (London) New Biol. 243:170The results of this study are compatible with 172. two recent independent studies on the in vitro 9. Lindahl, L. 1975. Intermediate and time kinetics of the reconstitution of ribosomal subunits from prein vivo assembly of Escherichia coli ribosomes. J. cursor rRNA species. Mangiarotti et al. (12) Mol. Biol. 92:15-37. reported the reconstitution of a 30S subunit 10. McCarthy, B. J., R. J. Britten, and R. B. Roberts. 1962. The synthesis ofribosomes inE. coli. JIl. Synthesis of without the formation of an intermediate partiribosomal RNA. Biophys. J. 2:57-82. cle when they used precursor 16S rRNA and 11. Mangiarotti, G., D. Apirion, D. Schlewinger, and L. proteins from nascent ribosomes. In the second Silengo. 1968. Biosynthetic precursors of 30S and 505 ribosomal particles in Escherichia coli. Biochemistry study, Nikolaev et al. (16) reconstituted a 53S 7:456-472. particle by incubating the large precursor 30S 12. Mangiarotti, G., E. Turco, C. Perlo, and F. Altmda. rRNA with the complete mixture of 50S and 1975. Role ofprecursor 16S RNA in assembly of E. coli 30S proteins. If the 53S reconstituted particle is 30S ribosomes. Nature (London) 263:569-570. then incubated with ribonuclease HI, both 50S 13. Mangiarottl, G., E. Turco, A. Ponzetto, and F. Altruda. 1974. Precursor 16S RNA in active 30S riboand 30S precursor particles are produced. All of somes. Nature (London) 247:147-148. these precursor particles are thus formed with- 14. Nashimoto, H., W. Held, E. Kaldschmidt, and M. Noout any observed slower-moving intermediate. mura 1971. Structure and function of bacterial ribosomes. XII. Accumulation of 21S particles by some It is possible that the regions of the sucrose cold-sensitive mutants of Escherichia coli. J. Mol. gradients that contain the 5OS and 30S precurBiol. 62:121-138. sor particles reported here and by Lindahl (9) 15. Nierhaus, K., K. Bordasch, and H. Hon'an. 1973. Riboinclude not only the earliest stages of assembly somal proteins. XLII. In vivo assembly of Escherichia coli ribosomal proteins. J. Mol. Biol. 74:587but later stages as well. All of them can be 597. indistinguishable by sedimentation (i.e., they 16. Nikolsev, N., D. Glazier, and D. Schlesinger. 1975. still sediment at 50S and 30S) if the cell exCleavage by ribonuclease III of the complex of 30S tracts are produced by gentle means. The heterpre-ribosomal RNA and ribosomal proteins of Escherichaa coli. J. Mol. Biol. 94:3014304. ogeneity of the 50S and 30S precursor partiN., and D. Schlesinger. 1974. Binding of cles-and their intrinsic lack of compactness 17. Nikolnev, ribosomal proteins to 308 pre-ribosomal ribonucleic compared to mature subunits -would then be acid ofEscherichia coli. Biochemistry 13:4272-4278. revealed when bacterial cells are disrupted by 18. Nomura, M., and V. A. Erdmann. 1970. Reconstitution ofthe 505 ribosomal subunits from dissociated molecharsh means or through the use of a detergent.
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21.
22. 23.
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ular components. Nature (London) 228:744-748. Osawa, S., E. Otaka, T. Itoh, and T. Fukui. 1969. Biosynthesis of 50S ribosomal subunit in Escherichia coli. J. Mol. Biol. 40:321-351. Patterson, D., M. Weinstein, R. Nixon, and D. Gillepsie. 1970. Interaction of ribosomes and the cell envelope of Escherichia coli mediated by lysozyme. J. Bacteriol. 101:584-591. Roth, G. S., and L. Daneo-Moore. 1971. Intracellular location of ribosome biosynthesis in osmotically fragile forms of Streptococcus faecalis. Biochim. Biophys. Acta 240:575-587. Sypherd, P. S., R. E. Bryant, K. Dimmitt, and P. Fujisawa. 1974. Genetic control of ribosome assembly. J. Supramol. Struct. 2:166-177. Tai, P.-C., D. P. Kessler, and J. Ingraham. 1969. Coldsensitive mutations in Salmonella typhimurium which affect ribosomes synthesis. J. Bacteriol.
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24. Traub, P., and M. Nomura. 1969. Structure and function of Escherichia coli ribosomes. VI. Mechanism of assembly of 30S ribosomes studies in vitro. J. Mol. Biol. 40:391-413. 25. Tremblay, G. Y., M. J. Daniels, and M. Schaecter. 1969. Isolation of a cell membrane-DNA-nascent RNA complex from bacteria. J. Mol. Biol. 40:65-76. 26. Van-Dijk-Salkinoja, M. S., and R. J. Planta. 1970. Formation and life cycle of ribosomal subunits in Bacillus licheniformis. Arch. Biochem. Biophys. 141:477488. 27. Van-Dijk-Salkinoja, M. S., T. J. Stoof, and R. J. Planta. 1970. The distribution of polysomes, ribosomes, and ribosomal subunits in exponential-phase cells of Bacillus licheniformis. Eur. J. Biochem. 12:474-482. 28. Wachsman, J. T., and R. Storch. 1965. Propionateinduced lysis of protoplasts of Bacillus megaterium. J. Bacteriol. 80:600-606.
