J. Mol. Biol.

(1979) 135, 611-626

Protein Synthesis in Bacillus subtilis I. Hydrodynamics and in Vitro Functional Properties of Ribosomes from B. subiilis W168 WILLIAM

J. SHAw%ocKt

and JESSE C. RABINOWITZ

Department of Biochemistry University of California, Berkeley Berkeley, CA 94720 U.S.A. (Received

7 March

1979, and in revised form 20 August

1979)

On the basis of their sedimentation properties, the ribosomal particles in crude extracts of Bacillus subtilis W168 are characterized as pressure-sensitive couples, pressure-resistant couples, or non-associating subunits. Pressuresensitive couples dissociate into subunits, yielding a peak at) 60 S in the gradient profile, on sedimentation at high speed in t)he presence of 10 to 15 m&I-Mg2 + . Under the same conditions, pressure-resistant couples sediment’ at 70 S. Under certain conditions, pressure-resistant couples apparently aggregate, possibly in 70 S * 70 S dimers. Procedures are described for the isolation of pressure-sensitive couples from B. subtilis. The isolat,ed couples are shown by chemical fixation experiments to require approximately twice the Mg2+ concentration required by Escherichia coli couples to remain associated at atmospheric pressure. All t,hree types of B. subtdis ribosome incorporate amino acids into acidinsoluble material in the presence of B. subtilis cellular RNA, B. subtilis ribosomal salt wash fraction, and E. coEi post-ribosomal supernatant. Overall incorporation, dependence on added RNA, and dependence on salt wash fract,ion arc greatest with prcssurc-sensitive couples. The products of protein synthesis in vitro stimulated by total B. subtilis RNA appear to be a low molecular weight subset of the proteins synthesized most abundantly in vivo. Incubation of pressure-sensitive couples with cellular RNA from B. subtdis, fMet-tRNAyet, ribosomal salt wash fraction and GTP results in their conversion to pressureresistant, couples, with concomitant and stoichiometric binding of fMet-tRNA to the 70 S species. It is concluded that in B. subtilis as in E. coli, pressure-sensitive while pressure-resistant couples are “complexed” with couples are “vacant”, messenger RNA. fMet-tRNA-bearing complexed couples are interpreted as initiation comploxcs in which ribosomes have bound mRNA, presumably at, initiation sites. Their formation in vitro is strictly dependent on RNA, salt wash fraction and fMet-tRNA when vacant ribosomal couples are used.

1. Introduction Recent reports of selective translation of natural messenger RNAs by ribosomes from gram-positive bacteria (Legault-Demare & Chambliss, 1976; Stallcup et al., 1976; t Present address: England.

M.R.C. Lehoratory

of Molecular

Biology,

Hills Road, Cambridge,

CB2 2QH,

611

0141-076S/79/350611-16

$02.00/O

0 1979 Academic

Press Inc.

(London)

Ltd.

612

W.

J. SHARROC’K

.4ND

J. C‘. 1~~AF3I~TOWITZ

Stallcup & Rabinowitz, 1973a,b; Lodish, 1969, 1970: &eitz. 1969) as well as tht, growing interest in the sporulating bacilli as model systems for t hc study of developmental regulation have made more detailed informabion on protein svnt hrsis in Bacillus subtilis desirable. Clearly the rigorous analysis of such phenomena as selective mRr\‘A branslation and possible translational control during sporulat,ion will require more precisely defined ~TLz’ikro systems than have previously bcien availabhl In particular, it is desirable that ribosomcs be obtaintbcl from gram-positive bacteria. free of endogenous mRNA. The qu&ion of specificity is. in large part. one of intcaractions between ribosomes from gram-positive bacteria and mRNAs from gramnegative sources. the former being apparently unable to tjranslato the latter. ‘l’h(~ presence of endogenous gram-posit.& mRNA would serioutily complicat,r the> int(Lrpretation of experiments in which very weak interactions might be expt~ctcd bet \j’t’t’tl the traditJional procedures messagct. Furthermortb. ribosomes and an untranslated used to render ribosomes frrlcl of associated prot,r:ins anal RSAt: (e.g. rxposur(~ to buffers of high ionic st,rength and preincubation) nlight br c~xpcct~ed to rE,suIt in depletion or degradation of important’ rif)osoma.l components. ‘I’has. in thest, st uclivi efforts have been directed at t)he isolation from B. ~suhtilis of a h~tlrodgl~a~ni~all~. uniform and highlp active population of ribosomat couplr~s. without rtwrtinp to high salt washing or preincubation.

2. Materials and Methods (a) Hacterial

strains

l)txpt of Bacteriology an to each sample contained 1 A 260 unit in 0.050 ml. These solutions were allowrd at 0°C for 10 min. Then 0.050 ml of 2.Oqb glutaraldehyde in t,he same hnffer bo each sample. The reactions were mixed, left at> 0°C for 5 min, and layrrr,d on a,b 56,000 revs/min sucrose gradients at 13 mm-Mg 2 + . Gradients were centrifuged and scanned for absorbance at, 260 nm. (j)

Labeling

of in vivo

translations

products

in B. subtilis

TV168

For labeling of in &JO translation products, B. suhtilis TVas grown in t.hc defined medium of Shub (1975). Labeling was carried out essentially as descrihod by Carrascosa et rsb. (1976) except that] [3H]valine was used as the limiting amino acid at, a specific activity ol 2.5 Ci mmol-I. A 5-ml culture, under forced aeration, was labeled for 5 min at 37°C. (k)

Protein

synthesis

in vitro

and

analysis

of pducts

by St~allcup &, Protein synthesis ?:r~ vitro was performed essentially as &scribrti Rabinowitz (1973a), except that in most, experiment,s (as indicat.etl in t,he Figure legends) the total reaction volume was 60~1 and each assay contained only 1 A260 unit of ribosomc,s. Unless otherwise specified, prot)ein synthesis in vitro w&s carried out at 11 to 12 mM-Mg” + compounds), 50 to (total concn, ignoring complexing of Mg2+ with phosphorylat.etl salt wash protein/ml. Ribosomal salt, wash 100 mM-NH, + , and O-5 to I.0 mg ribosomal fraction was from the same bacterial species as the ribosome preparation in every cast‘. The source of supernatant (at 100,000 g) enzymes and factors was in all cases the S 150’1 fraction from E. coli MRE600, the preparation of which has been described (Sta,llcup et al.. 1976). Supernatant fraction was prepared from H. aubtilis W168 and was found to support incorporation into polypeptides electrophoretically simila,r to those synthesized in tht$ presence of E. coli S150T. The efficiency of incorporation, however, was lower wit,h t hc B. 8ubtiZi.s fraction, possibly because of higher levels of degrsdativc? enzymes. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was performed a.s dPscrilj(~~l et al. (1976), except that gels werr of uniform sc*ryIamide conccntrat.iorl by Stallcup (1304) and were run at. 30 to 35 mA (175 to 260 V) only until the> dyfx marker reached t.hc* end of the gel. Fluorographic analysis of dried gels was performed as described by Bonncl & Laskey (1974). (1) Preparation

of ribosowaal

salt u?aah,fractiom

Salt wash fraction from E. coli MREGOO was prepared either as tlescribed by Stallcup & Rabinowitz (1973a) or by t,he method described below for wash fraction from R. subtdis W168. Salt wash fractions prepared from E. coli by the 2 m&hods were functionally indistinguishable. For preparat,ions of ribosomal salt wash fraction from H. subtilis M:168, 2 volumt~ of 2~ crude extract were underlaid with 1 volume of 15q;, glycerol in the extract. buffer and centrifuged for I6 h at, 64,000 g. The pellets were taken up in buffer without phenylmethylsulfonyl fluoride, adjusted to 1.0 M-NH,Cl by t,he a.ddit,ion of buffer containing NH,Cl at 1.2 M and stirred at. 0°C for 60 min. This suspension was then centrifugetl as above, over a 20”: glycerol cushion. To the supernaeant (salt wash) fraction, solid ammoniumsulfate was added to 850,b saturation, and the resulting precipitate was dissolvrti in a small volume of 20 m&f-Tris*HCl (pH 7*8), 20 mivr-NH,Cl, 2 mM-magnesium acct,at,r, 7 mM-2-mercaptoethanol, 50/b glycerol. After dialysis against the same buffer, t)hr concentrated fraction was clarified by centrifugation. (m) Isolation

of bacterial

cellular

HI\i-4

For most, experiments, total cellular RNA was extracted essentially as described 1973a) and I3. subtilis (Stallcup et nl.. previously for E. coli (Stallcup & Rabinowitz, 1976). This method includes a rapid chill and subsequent, digestion by lysozyme in t)ufftst at high cell density and 37”C, and employs neit,her st,erile glassware nor nuclease inhibitors. A second method, designed to minimize disruption of met,abolism prior t’o cell lysis and to reduce nucleolytic degradation of RNA during its isolation, was developed in the courst’

B. SUBTILIS

RIBOSOMES

615

of this work but did not significantly alter the pattern of in vitro translation products obtained. For use in fMet-tRNA binding experiments, RNA was fractionated in the zonal rotor as follows. The RNA from 2 cultures of 1 1 each was dissolved in 20 ml of 50 m&r-sodium acetate (pH 5), 100 mM-NaCl, 1 mm-Mgcl,, 2.5 M sucrose, and applied to a 400-ml 100/b to 20% sucrose gradient in the same buffer. The rotor was centrifuged for 5 h at 48,000 revs/ min. Fractions excluding the 5 S/tRNA peak were pooled and precipitated with ‘2 volumes of ethanol. The precipitate was recovered by centrifugation, dissolved in a small volume of sterile water, and stored at -70°C. (n) Preparation

of radioactively

labeled fMet-tRNA,Met

Purified tRNAyst (1400 pmol methionine accepted/A,,, unit) was charged with Q4C]methionine (250 mCi/mmol) or L-[3H]methionine (36 Ci/mmol) in a reaction containing 100 mM-Tris*HCl (pH 7*7), 15 mM-MgCl,, 20 mM-2-mercaptoethanol, 5 mM-ATP, 10 mM-ammonium acetate, 0.03 mM-lo-formyltetrahydrofolate (prepared as and 5.6 mg E. coli MRE600 described by Samuel et al., 1970), 0.10 mM total L-methionine, S15OT protein/ml (prepared as described by Stallcup et al., 1976). fMet-tRNAyet was recovered as described by Stallcup & Rabinowitz (1973a). On t,he basis of the known specific activity of the labeled methionine, aminoacylation of tQe tRNA was essentially complete. A separate experiment with 10-[14C]fornlyltetrahydrofolate established that the amount of S150T protein used here supplied a saturating amount of transformylase. (0) Analysis

of initiation

complex formation

For analysis of initiation complex formation, reactions contained 1 A,,, unit of ribosomes in 0.10 ml. Other components were 20 mM-TrisHCl (pH 7.7), 100 mm-NH,Cl, 5 mM-magnesium acetate, 1 mM-dithiothreitol, 1 mM-GTP, 0.4 PM labeled fMet-tRNAyst, 0.5 to 1.0 mg of B. aubtilis salt wash fraction/ml, where indicated, 0.1 to 0.2 mg E. coli salt wash/ml, where indicated, and messenger RNA, as indicated. Reactions were assembled at O”C, with the fMet-tRNA and mRNA, if any, added last. Reactions containing natural RNA were incubated at 37°C for 10 min, then applied immediately to sucrose gradients at 13 m&i-Mg2+ (for B. subtiZis ribosomes) or 5 to 7 mM-Mg2+ (for E. coZi), and centrifuged at 56,000 revs/min for 90 min. Reactions containing poly(A,G,U) were incubated for 5 min at 30°C and analyzed similarly. Absorbance scans were obtained as described in section (f), above, except that the outlet from the flow-cell terminated over an automatic fraction collector mounted directly under the spectrophotometer. Fractions of approximately 0.10 ml each were collected in 1.7 cm x 5.2 cm glass scintillation vials, diluted with 0.40 ml of water, and counted in 5.0 ml Omnifluor/toluene/Triton X-100 (Tiemeier & Milman, 1972).

3. Results (a) Hydrodynamics of ribosomes from B. subtilis

W168

When crude (S-30) extracts from B. subtilis W168 are sedimented through analytical sucrose gradients and scanned for absorbance as described in Materials and Methods, section (f), the characteristics of the absorbance profiles are found to Figure 1 shows a series of profiles vary with both rotor speed and Mg2 + concentration. produced at 56,000 revs/min (300,000 g at rave) and various Mg2 + concentrations. Material that sediments at 70 S in the presence of 30 mM-Mg2+ migrates with progressively lower apparent sedimentation coefficients as the Mg2 + concentration is reduced. profiles exhibit a new peak, with an apparent At. intermediate Mg2 + concentrations, sedimentation coefficient of about 60 S. At 6 mM-Mg 2 + , this peak is not observed and the areas under the 30 S and 50 S peaks are increased over their areas in the profiles at high Mg2 + . The same effect is observed at lower rotor speeds, except that lower are required to produce the appearance of a 60 S peak. Mg2+ concentrations

616

W.

J.

SHARROCK

AND

J.

C.

BABINOWITZ

Frc. 1. Sedimentation analysis of crude (S-30) extract from B. subtilis W 16~ at 56,000 revs/mill (300,000 g) for 90 min at various Mg2+ concentrations as noted. Gradients and samples WFIY’ prepared as described in Materials and Methods, section (f). Samples contained I.1 A,,, units ill 0.075 ml. Extract was prepared from a 1 1 culture. Absorbance scans were performed by upww(l displacement through a Gilford flow-cell. Sedimentation is from left to right. Maximum peak height represents an absorbanre of approximately 0.40.

These results are consistent with a shift in the ribosomal subunit associat,ion equilibrium towards dissociation under conditions of high hvdrostatic pressure and towards association in the presence of high Mg2+ concentrations. The 60 S peak it) the absorbance profile consists of 50 S ribosomal subunitIs that, have sedimented for part of the gradient length as half of a 70 S couple. 30 S subunits arising from t,h(b same pressure-induced dissociation are found in the 50 S region of the gradient (e.g. Fig. 5(a)) but are generally obscured by free 50 A subunits in analyses of crude extracts. The persist,ence of a peak at 70 S even undclr conditions that shift the bulk of t,he material to the subunit) positions indicates t#hat some ribosomal couples art’ not subject to pressure-induced dissociation. The couples that’ dissociate (i.e. product> a 60 S peak) at’ 56,000 revs/min and 13 mM-Mg2+ are referred t,o hereinaft,er as pressure-sensit,ive, while . t~hose bhat sediment, at 70 8 under these conditions arc’ referred to as pressure-resistant,. In order t,o determine whether species larger t’han 70 8 are present) in B. suhtili.v extracts, samples were sedimented at various Mg2+ concentrat’ions as in Figure I. but for only 50 minutes (Fig. 2). These profiles indicate that several species with apparent sedimentat,ion coefficient,s greater than 70 S are present. However. tht most prominent of these, while it, is observed to sediment, at 90 t,o 100 S at 15 mM-Mg” + sediments more slowly at) lower Mg2+ concent,rations, final1.v oosedimenting with 70 8 couples at 7 mM-Mg2+. In Figure 3. the apparent sedimentation coefficient of this species is shown to be dependent on the duration of centrifugat,ion as well as t,hcb Mg2+ concentration. Material that, constitutes a dist’inct peak at 90 to 100 S in a 40-minute spin appears as a shoulder on the 70 S peak in a 5O-minute spin and cosediment,s with 70 S couples in a ‘IO-minutle centrifugation. A QO-minute spin, as

B. SUBTILE3

RIBOSOMES

l(b)

e)

617

“’

1

. Sedimentation

coefficient (S)

FIG. 2. Sedimentation analysis of crude (S-30) extract from B. subt& W168 at 56,000 revs/min (300,000 g) for 50 min at (a) 15 miw, (b) 13 mrx, (c), I1 mM, (d) 9 mM, (e) 7 mM, and (f) 5 mM-Mg,+. Extract was prepared from a 12-1 culture. Samples contained 1.7 A,,, units in 0.075 ml. Absorbance scans were obtained by downward displacement through a MICO flow-cell. Sedimentation is from right to left.

expected from the experiments shown in Figure 1, results in the appearance of a new peak at 60 S. Preparative sedimentations described in the next section demonstrate that the peak above 70 S in low-speed gradients is an aggregate form of pressureresistant couples possibly 70 S. 70 S dimers. (b) Isolation of pressure-sensitive ribosomal cou$es from B. subtilis W168 Preparative centrifugation of crude extracts yields profiles similar in most respect,s t.o those obtained with small analytical gradients. Figure 4 shows the fractionation of an extract under conditions that do not induce dissociation of pressure-sensitive couples: 13 mM-Mg2+ and 35,000 revs/min (77,000g at, rave). As in t’he anal”ytical gradient,s of Figures 2 and 3, a peak with an apparent sedimentation coefficient greater than 70 S is observed. By pooling the fractions as indicated in Figure 4, and centrifuging samples of the concentrated pools through analytical gradients at high speed (profiles not shown), it was determined t)hat pressure-sensitive couples are segregated preferentially in the 70 S region (pools A and B), whereas the more rapidly sedimenting peak (pool C) consists largely of pressure-resistant couples. Thus absorbance peaks above and below the 70 S position correspond, respectively, to two different populations of ribosomes. Pressure-resistant couples sediment more rapidly

7

Sedlmentatlon

r-

coefficient

(S)

Fm. 3. Sedimentation analysis of crude (S-30) extracat from B. scthtilia WI 6X at I3 m~-Jly~’ and 56,000 revs/min (300,000 g) for (a) 40 min, (h) 50 min. (c.) 70 min. and (d) 90 min. Extract, was units in 0,075 ml. Absorbance scans \~erv prepared from a 12.1 culture. Samples contained I.7 A,,, Srtiimrnt,atio~~ is from right tc, obtained by downward displarement, through a MICO fl oxv-wll. left.

than 70 S under conditions of t,he zonal centrifugativn show in Figure 1, probabl?. by aggregating in 70 S.70 S dimers. Pressure-senskive couples, on the other hand, do not aggregate under these conditions. Further purification of pressure-sensitiw couples map be achieved by centrifugat,ion of material sitnilar to pools A and 33 of

0

100

200

300

Gradlent displaced

400 (ml)

&a. 4. Preparative density-gradient centrifugation at’ it wutk (8-30) cxtraot from B. S&~&Y W168 in the Ti-14 zonal rotor at 13 m~-i%lg~+ and 35,000 rrvs/min (77,000 g) for 180 min. Sucrow concentration ( ) is that programmed into the pump; in practiw. houndaries are diffuse. Thv was pi-vparrrl fwm a 12-l cukurr. Srdimt~ntasample contained 3200 A,,, units in 15 ml. Extraotj tion is from left to right.

B. SUBTZLIS RIBOSOMES

619

Figure 4 through an uncushioned 10% to 20% gradient at 4 mM-Mg2+ and 48,000 revs/min (profile not shown). Under these conditions, pressure-sensitive couple8 are dissociated into subunits. Fractions corresponding to the 30 S to 50 S region are pooled, yielding a preparation that is virtually free of pressure-resistant couples. This fract,ion, however, contains relatively large amounts of non-associating subunits, probably because of deleterious effect8 of the preparative sedimentation at low Mg2 + concentration. Pressure-sensitive Couple8 may be fractionated away from these non-associating subunits by centrifugation through an uncushioned gradient at’ 13 mM-Mga + and 35,000 revs/min (profile not shown). The final preparation of pressuresensitive couple8 is characterized by the absorbance profiles, from analVytical gradients at both high and low speed, shown in Figure 5.

Sedlmentotlon

cceffuent

(S) (b)

(a)

Fra. 5. Sedimentation analysis of final preparation of pressure-sensitive ribosomal couples from B. s&i& W168. Centrifugation was for (a) 90 min at 56,000 revs/min or (b) for 330 min at 30,000 revs/min at 13 mM-Mg,+. Samples contained 0.76 Azeo unit in 0.075 ml. Absorbance scans were obtained by downward displacement through a MICO flow-cell. Sedimentation is from right to left.

(c) Subunit association equilibrium of B. subtilis W168 pressure-sensitive couples at atmospheric pressure

Pressure-sensitive ribosomal couples from B. subtilis and E. coli were fixed with glutaraldehyde at various Mg2 + concentrations and analyzed by density-gradient, sedimentation as described in Materials and Methods, section (i). From each profile. the areas (A) under the 70 S, 50 S and 30 S peak8 were calculated and the ratio A 70s Asos + &OS + 40s

wa8 taken as the mass fraction of 70 S couples at equilibrium. The results are shown in Figure 6, with a curve obtained by Wishnia et al. (1975) for E. co& using lightscattering techniques, shown for comparison (broken line). It is clear that subunit association at atmospheric pressure is substantially more dependent on Mgzt in B. subtilis than in E. coli. The E. coli system is 50% couples by mass at about’ 2 mM-Mg2+, while B. subtilis ribosomes require approximately 4 mM-Mg2+ for 50% association. Between 4 and 5 mM-Mg 2+, E. coli ribosomes are virtually completely associated, while only 60 to 70% of the B. subtilis ribosome population is found as couples. This is consistent with the observation that pressure-induced dissociation of sensitive B. subtilis couple8 produces a 60 S peak in gradient profiles at high speed and 13 mM-Mg2+ (e.g. Fig. 3(d)), while the Mg2+ concentration must be reduced tjo 6 mM t,o produce the same effect with E. coli ribosomes (No11et aZ., 1973a).

620

W.

J.

SHARROCK

AND

J.

C.

HABlNOWITZ

Pm. 6. Chemical fixation analysis of ribosomal subunit’ association equilibria at various Jig” ’ concentrations and atmospheric pressure. Pressure-sensitive couples from B. subtdis ( -I j .-j ) were fixed with glutaraldehyde at the Mg2+ roncmtrations and E. coli MREGOO (-O-e-) indicated, and analyzed as described in Materials and Methods, section (i). The broken lint is the curve obtained by Wishnia et al. (1975) for E:. coli ribosomes, using light-scattering mcasmvmt,nts.

((1) Incorporation of amino cwids i,rlto polypeptidrs by ribosomes from B. subtilis u’l6X Figure 7(a) shows the amino acid incorporation activities in/ Gtro oft he three t,ypw of B. subtilis ribosomes defined by hydrodynamic properties in the preceding sections, in response to B. subtiEs cellular RNA. Pressure-sensitive couples produce the high& levels of incorporation and exhibit the lowest endogenous (-RNA) activiby, t,ypicall~ responding to added RNA with a 20-fold increase in incorporation. Figure 7(b) shows the dependence of incorporation on addition of the ribosomal salt wash fract,ion to tht: I

,

I

I

II

40

2.0 [#&A]

I

(A, (a)

units)

0u [Protein]

(pg) (b)

FIG. 7. Dependence of amino acid incorporation its v&o by B. ,s&tili~ I)ressure-srnsitivr, couples (o), pressure-resistant couples (A), and non-associating subunits ( V) on (a) added B. subtilis RNA and (b) B. subtilis ribosomal salt wash fraction. Pressure-resistant couples and non-associating subunits were recovered from fractions of preparative sucrose density-gradic,nt,s similar to that shown in Figure 4. Reactions in (b) contained 2.3 ,4 260 units of B. subtilis RNA. All reactions were 0.060 ml in volume and contained I Azso unit of ribosomes. Other components of the reaction mixture were as given in Materials and Methods, section (k).

B. SUBTILIS

621

RIBOSOMES

incubation. Non-associating subunits exhibit considerably more activity than does either type of 70 S couple in the absence of the wash fraction, suggesting that they carry bound initiation factors or other components of the salt wash fraction. In order to determine whether the natural RNA-stimulated amino acid incorporat,ion shown in Figure 7 reflects the synthesis of physiologically significant protein products, the products of reactions in vitro were resolved by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and compared with the proteins labeled in vivo by a five-minute pulse of [3H]valine. Figure 8 shows that nearly all of the in vitro products correspond in electrophoretic mobility to bands in the in vivo pattern. In the region of the gel corresponding to molecular weights from about 20,000 to about 30,000, several heavily labeled bands and many lightly labeled bands appear at similar relative intensities in both in vitro and in vivo patterns. Shorter exposures (not shown) demonstrate that the low molecular weight region of the in vitro pattern. which is overexposed in Figure 8, contains the same three bands as are observed in not shown here, RNA preparations from the in vivo pattern. In other experiments B. subtilis were translated by E. coli vacant couples, yielding patterns of products

(a)

(b)

(cl

I-

67,000

t

35,000

t

25,000

f-

14,000

Cd)

FIG. 8. Electrophoretic/fluorographic analysis of products of amino acid incorporation reactions containing B. subtilis W168 pressure-sensitive couples (a), pressure-resistant couples (c), and nonassociating subunits (d), compared with proteins labeled in viwo in B. subtilis W168 by a R-min pulse of [3H]valine (b). In vitro reactions were 0.060 ml in volume and contained 2.2 A,,, units of B. subtilis RNA. Proteins labeled in &JO were obtained as described in Materials and Methods, section (j). Protein synthesis in vitro was conducted as described in Materials and Methods, section (k). Samples for electrophoresis contained (a) 11,600, (b) 10,500, (c) 10,800, and (d) 12,000 3H cts/min in acid-insoluble material. The fluorogram was exposed for 12 days. >lolecular weight markers were bovine serum albumin (67,000), rabbit muscle glyceraldehydc 3-phosphat)e dehydrogenase (35,000), bovine pancreas chymotrypsinogen (25,000), and egg-white lysozyme (14,000).

622

W.

d.

SHAHHOCK

AND

J.

C!. RARlNO\\‘IT%

virtually identical t,o those shown in .Figurr 8 (a). (c) and (d). Thus: translation of total cellular RNA from B. subtdis in vitro appears to yield products that oonstituttl a low molecular weight subset of the proteins synthesized most abundantly i/r. Vito. (e) Formation

of initiation

complexes by pressure-sensiti,ue vhsonral couple.~ W16X

from B. subtilis

Figure 9 shows the profiles obtained on sedimcntstjion analysis of incubations containing B. .subtiZis pressure-set&k couples and various combinations of‘ U. :I ,~ubsubtiZis REA, B. subtilis ribosomal wash fraction. QTP and fMet-tRNA. stantial proportion of ribosomal couples acquires pressurc~-resista,rr~(, and binds fMet-t,RNA when all four of the latter components are present in a,ddition to ribosomes (Fig. 9(f)). Omission of RNA. fMet-tRP\‘A, or salt wash fraction abolishcls formation of 70 S complexes complet,ely (Fig. 9(b). (c) and (cl)). It has been ShlJ\,W if1 E. coli (see Discussion, section (a)) that, pressure-resistant ~o~qks arc’ “complexcd“ couples, i.e. have bound mRNA. Pressure-sensit,ive couples do not carry mRNA and are termed vacant couples. The results shown in Figure 9 are consist.& with t 11,~

Sedimentation

coefficient

(S)

FIG. 9. Format,ion of initiation con~plexes by pre~islr~c~-scnsiti~t, ribosomltl couples from H. subtilis W168. Incubations and analyses were as desrrihrd in Materials and Methods, section (o), with components omitted as follows: (a) salt wash fraction and RNA; (b) RNA: (0) fillettRNA; (d) salt wash fraction; (e) GTP; and (f) none (complete reaction). &actions (c) through (f) contained 2.6 Azea units of B. subtilis RNA. Broken lines in (c) through (f) indicate absorbanrc recorded at twice the indicated scale; ( -) absorbencc at, 260 nn~; -,?; ---’ ,I --. fMet.tR~NA~~.

B.SUBTIL1SRIBOSOMES

623

application of the terms vacant and complexed to pressure-sensitive and pressureresistant couples, respectively, in B. subtilis as in E. coli. Taking a value of 23 pmoles per A,,,, unit, as a compromise among the various data reported for the molar extinction coefficient of E. coli ribosomes (Hill et al., 1969), and assuming that the peaks in the absorbance profile represent simple symmetrical zones, the molar ratio of fMet-t,RNA to ribosomal couples in Figure 9(f) is calculated to be 1.1. This stoichiometry, wit’h the virtually complete dependence of formation of fMet-tRNA-bearing complexed couples on the salt wash fraction, argues strongly that these species are initiation complexes. Omission of GTP, while substantially reducing the binding of fMet-tRNA in 70 S complexes, appears to have less effect on the formation of complexed couples (Fig. 9(e)). It is of interest that addition of RNA appears to have a dissociation effect on ribosomal couples (Fig. 9(d) versus (a)). This suggests that RNA may be binding to one or both subunits, even when the salt wash fraction is not present, and no binding of fMet-tRNA to ribosomal species is apparent. When the synthetic polynucleotide poly(A,G,U) is substituted for natural RNA, extensive formation of fMet-tRNA-bearing complexed couples is also observed (data not shown). With the synthetic polynucleotide, however, the stoichiometry of Mert-tRNA binding is consistently only 0.8 to 0.9 pmoles per pmole of 70 S couple. f Futhermore, massive formation of complexed couples, accompanied by bizarre fMet-tRNA binding patterns, occurs when the ribosomal salt wash fraction is omitted from incubations containing poly(A,G,U). These results suggest strongly that poly(A,G,U) cannot be considered a good analog of natural mRNA in detailed studies of RNA binding and initiation. Pressure-resistant couples and non-associating subunits also form fMet-tRNAhearing 70 S complexes when incubated under the conditions of the initiation assay (dat#a not’ shown). However, it is observed that pressure-resistant couples bind substantial amounts of fMet-tRNA even in the absence of added RNA, while nonassociating subunits form 70 S complexes in the absence of salt wash fraction, These results are consistent with those of Figure 7 and reinforce the conclusion that pressureresistant couples are “complexed” with mRNA fragment,s, while non-associating subunit,s apparently carry components of the salt wash fraction. 4.

Discussion

(a) Pressure-induced dissociation of B. subtilis ribosomes The effects of hydrostatic pressure on the ribosomal subunit association equilibrium were first noted in work with eukaryotic ribosomes (Infante & Baierlein, 1971). Centrifugation at high speed was observed to produce peaks in the absorbance profile that did not correspond to either ribosomal couples or isolated subunits. Most conspicuously, a peak appeared between the positions corresponding to couples and large subunits, respectively. It was concluded that hydrostatic pressure, which increases as a composite function of the height of the fluid column above a given position, the density of the fluid, and the centrifugal force at that position, could shift tJhe subunit association equilibrium towards dissociation. No11 and his colleagues have shown that E. coli ribosomes can be characterized as vacant or complexed according to their sensitivity to pressure-induced dissociation (No11 et al., 1973a). It was observed that a fraction of the ribosomal couples from rapidly chilled cells continued to sediment with a coefficient of 70 S even under

024

IV. J. SHARROCK

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J. (!. l different, manner than do t,he mRh’As of gram-neyat’ive bacteria PJIC~ as E. coli is treated further in the accompanying paper. WC thank Cheryl Murray for export t,echnicul assistance. M’c, thank Mr Donald Healb. for expert) assistance in the design and manufacture of the mixing chambrr for the gradient pump. We thank M. Griffith of Beckman Instruments for advice on the use of the zonal rot,or. This work was supported by research grant AM02109 (to J. C. K.) and research training grant TOl/GM0031-19 (to t>hr Department of Biochemist.ry) from t,hc National Instit,utes of Health. U.S.A.. REFERENCES Bonner, W. M. & Laskey, H. A. (1974). Eur. ./. f&-hem. 46, 8388. Carrascosa, J. L., Camacho, A., Morcno, F., Jimenoz. F., Mellado. 1~. I’., \‘inucia, E. & Salas, M. (1976). Eur. J. Biochem. 66, 229-241. Das, H. K. dt Goldstein, A. (1968). ./. ~WoZ. B1:oZ. 31, 209 226. Friedman, H., Lu, P. & Rich, A. (1969). Nature (lmdon), 223, 909 ~913. Godefroy-Colburn, T., Wolfe, A. D., Dondon, .J., (irnnhcrg-Manago. M., Dcssrn. I’. k Pantaloni, D. (1975). J. Mol. Hiol. 94, 461 478. Hill, W. E., Rossetti, G. P. & Van Holde, K. E. (1969). .1. Jlol. Biol. 44, 263.-277. Infants, A. A. & Baierlein, R. (1971). Proc. Nat. Acarl. Sci.. I’.S.A. 68, 1780~-17X5. Legault-Demare, L. & Chambliss, G. H. (1974). J. Bactwiol. 120, 1300-1307. Legault-Demare, L. & Chambliss, C. H. (1976). :LloZ. Gen. &net. 142, 277 285. Lodish, H. F. (1969). Nature (London), 224, 867~-870. Lodish, H. F. (1970). Nature (London), 226, 705 707. McCarty, K. S., Stafford, D. & Brown, 0. (1968). Awal. Biochem. 24, 314 ~329. Noll, M. & Nob, H. (1974). ,I. &.!oZ. B&Z. 90, 2377251. Noll, M., Hapke, B., Schreier, M. H. Dt Noll, H. (1973a). J. .IZol. l&l. 75, 281l294. Noll, M., Hapke, B. & Noll, H. (1973b). J. MoZ. BioZ. 80, 519- 529. Samuel, C. E., D’Ari, L. & Rabinowitz, J. C. (1970). ,I. RioZ. Chem. 245, 5115~ 5121. Shub, D. A. (1975). NloZ. Cen. Genet. 137, 171-180. Spizizen, J. (1958). I’roc. Nat. Acad. Ski., IT.S.A. 44, 1072 1078. Stallcup, M. R. & Rabinowitz, J. C. (1973a). J. BioZ. Chem. 248, 3209-3.215. Stallcup, M. R. 62 Rabinowitz, J. C. (19735). ./. BioZ. Chem. 248, 3216-3219. Stallcup, M. R., Sharrock, W. J. & Rabinowitz, J. C. (1976).J. BioZ. Cherra. 251, 249% 2510. Steitz, J. A. (1969). Nature (London), 224, 957.-964. Subramanian, A. R. & Davis, B. D. (1970). Nature (/m&m), 228, 127% 1275. Tiemeier, D. C. & Milman, C. (1972). J. Riol. Chem. 247, 2272-2277. Wishnia, A., Boussert, A., Graffe, M., Dessen, P. B (irunberg-Manapo, M. (1975). .1. :VoZ. BioZ. 93, 499-515.

Protein synthesis in Bacillus subtilis. I. Hydrodynamics and in vitro functional properties of ribosomes from B. subtilis W168.

J. Mol. Biol. (1979) 135, 611-626 Protein Synthesis in Bacillus subtilis I. Hydrodynamics and in Vitro Functional Properties of Ribosomes from B. su...
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