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Ribosomes in thiostrepton-resistant mutants of Bacillus megaterium lacking a single 50 S subunit protein
J. Mol. Biol.
(1979) 132, 235252
Ribosomes in Thiostrepton-resistant Mutants of Bacillus megaterium Lacking a Single 50 S Subunit Protein ERIC CUNDLIFFE, PETER DIXON, MICHAEL STARK Department
of Biochemistry, University Leicester, England
of Leicester
GEORGST~FFLER, RENATE EHRLICH,MARINA ST~FFLER-MEILICKE Max-Plan&-Institut
fiir Molek,ulare Berlin-Dahlem,
Gelzetik, Ahteilung
Wittm,ann
Germany
AND MICHAELCANNON Department of Biochemistry King’s College, University of London London, England (Received 28 December 1978, and in revised ,form 11 April
197’9)
A protein required for the binding of thiostrepton to ribosomes of Bacillus megaterium has been purified and furt,her characterized by immunological techniques. This protein, which does not bind the drug off tllc ribosome, is serologically-homologous to Escherichia coli ribosomal protein Ll 1 and is designated BM-Lll. Ribosomes from certain thiostrepton-resistant mutants of R. megaterium appear to be totally devoid of protein BM-Lll as judged by modified immunoelectrophoresis. Such ribosomes are significantly less sensitive than those> from wild-type organisms to the action of thiostrepton in vitro but retain subst,ant,ial protein synthetic activity. Re-addition of protein BM-Lll to ribosomrs from the mutants restores them to wild-type levels of activity and thiostrepton sensitivity. Thus ribosomal protein BM-Lll is involved not only in binding thiostrepton but also in determining tile thiostrepton phenotype.
1. Introduction binds to bacterial ribosomes and prevents their functional interaction with various protein factors involved in the initiation, elongation or termination stages of polypeptide synthesis (for a review see Cundliffe, 1979). Consequently. hydrolysis of GTP, dependent upon the binding of such factors t’o ribosomes, is inhibited in vitro. The observation that the closely related antibiotics thiostrepton and siomycin are able to inhibit functions involving both the elongation factors EF-Tu and EF-G led to the idea that ribosomes possess a single site which is utilised by these factors alternately and which, together with either factor, const,itut’es a GTPase centre (Cundliffe, 1971: Modolell et al., 1971). Rihosomes of Escherichia coli possess a single binding site for thiostrepton, located 235 4‘ 1979Alcadenlic Prow Inc. (I~~nrlon) Ltcl. 00”“~ 283A/79/%20235-18 $02.00/o
Thiostrepton
236
E. C’~TN1~I,Il~FE
ET
AL.
on the 50 8 ribosomal subunit (Sopori & Lengyel. 1972: Highland it nd.. 1975~1,).tc, which the drug binds with high affinity. This site is disrupted why 50 S subunits it1.t’ exposed to 1 M-LiCI, thereby producing “I M-LiCI (~JI’O partjicl~~s”. I)ut (aan 1~. I’(‘rt (I/.. 197%). \ic’hct~ assembled IJ~ adding back ribosomal prot,ein IA I (Highlantl 1 M-Lick split proteins were treated with ant&ra raised against various purified ribosomal proteins, only immunoglobulin raised against protein I,1 1 abolishrtl thth ability of split proteins to restore to cores the abilit,y to hind thiostrepton. Xccordingly and as a result, of other experiments, it was concluded (Highland it ~1.. 197,%) that protein Lil is required for the binding of thiostrepton to riboxomes of E. co/i. Experiments involving the characterisation of antibiot,ic- binding sites by partial reconstitubion of ribosomes from core particles a,nd split proteins can br usefUy complement’ed by examination of mutationally altered riboaomex ohtaincd f’rom drug-resistant mutants and also by binding studies with aflinity analogurs of antibiotics. In t,his work we did not use t,hiostreptoll-I,esist,ant mutants of KF:.loli sinc*cs the wild-type of that, organism does not take up the drug. Mut,ants of E. c,o/i \vhich allow penetration of thiostrepton can only h otjtaintd hy tlrcs ww of’ tnutagws (Liou et ul., 1975). a procedure \vhich we wished to avoid. Xocordingly. w(& Ilavt* examined the ribosomal binding s&e for thiostrepton ill Racdllus /r,egaf~~irrnr ill1 organism which is sensitive to the drug. Here wt’ report that ribosumes of B. tttegrcterium contain a protein which is immunologically relat)cd to ribosomal protoitl LI I of E. ~4% and which is involved in the binding of thiostrepton to ribosomrs. 1n addition. we discuss the properties of the ribosomes of three spontnnr~ousl~~ arising mutant strains of B. vtz~egaterium which are resistant8 to tjhiostrrpton.
2. Materials and Methods (a) Selection
of mutart&
and maintena?rce
of strains
Mutants of 13. megateriwm KM we’re selected by plating the wild-typo on Irutrient :rgur containing thiostrepton and arose spontaneously with a frequrncy of approx. 1 ill 10y. The mutant designated MJl was selected on 3 pg thiostrepton per ml of nutrietlt agar whereas mutants PDl and PD14 were selected at 10 p*fs drug per ml. Mutants werct maintained on nutrient agar plates cont,aining 3 pg thiostrept,on per ml, tha minimull growth-inhibitory concentration for the wild-type under these conditions being appros. 0.1 pg drug per ml. In the absence of drug, all the mutants studied CIWC:gram \vith a nleat~ gelleratiotl tjltrrcL approx. 2.5 times great,er than that of the wild-t.ype. Accordingly, when growing t,lrv mutants in bulk, starter cultures cont,ained t,hiostrrpton although growtll was necessaril), continued in the absence of drug. Subsequent, examination revealed that thp rrrlttatltjs were quite stable in the absence of drug and that revertams to wild-type did not arise during bulk growth of mut)ant,s. (b)
Preparation
of ribosomal
core particles
ad
s&t
protein~s
Cells were broken by passing them t,wice t’hrough a chilled Frencll Press operated a~ approx. 12,000 lb/in2. During tllis procedure the concentzation of cell-paste was approx. 500 mg wet weight, of cells per ml in buffer containing 10 m&l-Tris-acet,ata (pH 7.6 at 2O”C), 10 mlvr-magnesium acetate, 50 mM-ammonium acetate. 3 mM-8-mercaptoet~ianol. 0.5 mM-EDTA. Deoxyribonuclease (5 pg/ml) was then added and after 5 min at 0°C: cell debris was removed by centrifugation at 30,000 g for 30 min. Ribosomes were their prt*cipitated by centrifugation in the Beckman Ti70 rotor at 50,000 revs/Knin for 6 11 and the, post-ribosomal supernatant (5100) was dialysedat) 0°C against, 3 x 100 vol. of ribosomc~ storayts storagca buffer (RS bluffer) buffer before being stored a,s samples at - i’O”(‘. Ribosnme
THIOSTREPTON-RESISTANT
RIBOSOMES
237
cont,ained 10 m&I-Tris.HCl (pH 7.6), 10 mM-MgCl,, 50 mM-NH&l, 3 mm-/3-mercaptoethanol. Crude ribosomes were resuspended in RS buffer and were washed once by layering thorn over RS buffer containing 40% ( w / v ) sucrose and centrifuging them for a minimum of 7 II at 50,000 revs/min in the Beckman Ti70 rotor. The resultant “sucrose-washed” ribosomes were resuspended in buffer containing 10 mM-Tris.HCl (pH 7*6), 30 mM-MgCl,, 1 &I-NH,Cl, 3 mM-P-mercaptoethanol and were layered over similar buffer containing 20:/, (w/v) sucrose. They were then centrifuged at, 50,000 revs/min for 5 h in the Beckman Ti70 rotor. This “high salt” washing procedure was carried out 3 times before ribosomos were resuspended in, and dialysed against, RS buffer prior to storage as samples at - 70°C’. Core particles were produced in buffer containing 10 m&I-Tris*HCl (pH 7.6), 1 rn>IMgCl,, 3 mM-8.mercaptoethanol together with LiCl (1 M, or 2 M), to which ribosomes were added at 2 mg/ml final concentration. After 17 h at O”C, core particles were collected by centrifugation at 45,000 revs/min for 6 h in the Beckman Ti75 rotor and were resuspended in, dialysed against, and stored at -70°C in, RS buffer. The LiCl extract which contained split proteins was concentrated by dialysis against RS buffer containing 207; (w/v) polyethylene glycol (PEG 6000) at 0°C. Following low-speed centrifugation, of approximately split proteins were st,ored in RS buffer at - 70°C at concentrations 4 nmol equivalents per ml. One nmol equivalent, of split proteins was defined as the amount obtjained from 1 nmol of ribosomes. (c) Assay for bindiny
of [35S]thiostre@on
to ribosomal
particles
to This was carried out as previously described (Dixon et al., 1975) in buffer identical that used for storage of ribosomes, cores and split proteins. Cores or ribosomes, usually 25 t,o 50 pmol in 50 ~1 RS buffer were mixed with 5 ~1 [35S]thiostrepton (60 pmol) dissolved in dimethylsulphoxide. After 10 min at room temperature 100 ~1 buffer was added and the whole incubation mixture applied to a column of Sepharose 6B or Bio-Gel Al.5 m (5 cm x 0.55 cm) equilibrated with RS buffer. Binding was determined by estimation of radioactivity in tile void volume by liquid-scintillation spect’rometry. Various preparations of [35S]tlliostrepton were used during the course of these experiments at specific radionativities in the range of 200 to 500 cts/miri per pmol. (d) Reconstitution
of ribosomal
particles
from
cores and split
proteins
This was carried out according to Highland et al. (19753). Cores were mixed with 1.5 to 2 excess equivalents of split proteins and kept at 0°C for 5 min and then at room tempc,ratllrc for a further 5 min. Radioactive thiostrepton was then added and its binding assayed as above. RS buffer was used throughout. (e) Chromatography
Split
of split
proteins
on carboxymethylcellulose
proteins
were obtained from 900 mg of NH,Cl-washed ribosomes of wild-type B. meyaterirur~ KM by extraction with I M-Lick. These “1 M-LiCl split proteins” were dialysed into 2qb (v/v) acet,ic acid and concent,rated to 20 ml final volume by dialysis against 276 acetic acid containing 20% (w/v) PEG 6000. Concentrated split proteins were then dialysed exllaustively against CM buffer (6 Al-urea, 30 rnM-methylamine, 1 m&Idithiothreitol, pH 5.6 with acetic acid). The 1 M-LiCl split proteins in CM bufl’er were loaded at room temperature onto a column (14 cm x 1 cm; bed vol. 11 ml) of carboxymethylcellulose (Whatman CM-23) equilibrated with CM buffer. The column was washed with 10 ml CM buffer and then developed using 100 ml of a 0 to 100 InM gradient of sodium acetate in CM buffer. Fractions (1 ml each) were collected and samples of each were analysed by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and were assayed for their ability to restore to 2 M-LiCl core particles the ability to bind [35S]thiostrepton. “Active fractions” were pooled (see Fig. 1). They were then desalted over Sephadex G25 (equilibrated with O.lOi, (v/v) acet,ic acid), lyophilized and re-dissolved in 1 ml CM buffer. (f) Chromatography
on Sephadex
GlOO
Following ion-exchange chromatography on carboxymethylcellulose, proteins were further fractionated on a column (100 cm x 1.5 cm) of Sephadex ($100 in CM buffer.
‘I’HIORTICEl’TON-RESIS’~.~~NT (rn) lmm~tnochemical
,methot/s
Atlt~isera to E. cull: ribosomal protein Lll were prepared (Stiiffler. 1974; St,iiffler 85 Wittmann, 197la,b). (II)
I’reparation
of antiserum
“:l!f
RIHOSOMl3S
to the H. Ineqterillrn
a~ld characterized
thiostrepton
as describvd
-hl:ndinq proteirl
A slletp (H80) was irnmutGzet1 \vitll a total alnount of 70 1~ of the protein, purified from R. m,eqaterium 70 S rihoxotnes. Tlrr prc&ein was dissolved in 3.5 ml of 8 M-urea alld stored a.t -40°C in 7 samples of 0.5 ml cacb. Prior to immunization each sample was diluted wit’h 2.0 ml of pllospllatc-bllffrrcd saline (PBS) anti t’he solution emulsified with 2.5 ml i+‘rctlrrtl’s complete adjlivant (lXfc0). Irrjcrtions were administered intramuscularl> atld s~lbcntatieo~lsly at 8 different, sites on days 0, !I. 15, 18. 22, 56 and 60; the animal n-a.s bled on days 0, 27. 33 and 64. Tlrr alttisera \vew incallbatcd at 56°C for 30 mitr to inactivate cotnplrmer~t. For some exprrimcnt~s irrnn~~no~l~~hnlills XVCI’(’ oorlwtlt~ratctl by precipitatiolr a1 40”~,, satrlratioll \vitll ammonitnn sldpllntv. Tlrv precipitates \+c>I’P dissolved it1 L’ES. dialysetl ansinst tllc* same brlfler alld stored at - 40°C’ or lyoplrilized and st)ored a,t 4 (‘. Dvtaiis of tlrr> spwificity of tlris arltiserllm ark prewtltc~
Experirncnts \vere performed (Olvzhterlorlv, 1RO8), with modifications, as tlecnssa~ry for tllc arlalysis of ribosomal protrirrs (Stiiffler & \Vittmanll. 1Wla.h: St)iiffler, 1974).
Tllis was according to Stiiffler (1967) u it h millor modificatiorrs (Zubke et al., 1977; Wierwrr et al., 1979; G. Stijfflrr & K. Ehrlicl~. unpublished dnt,a). Thrco buffers were used: bttffer ,>1 contained 0.12 nz-bls-Tris ([t~is-(2-lrydrox~~~tl~~~l)irrrir~o-tris-(l~ytlroxymetl-~yl)mctlrane]), 0.12 N-MN (2-1~-lnorplrolirro ctllatrv sr~lpl~on~c acid). 0.05~1.2.~nercaptoetllanolitlld 8 ai-nrea. Buffer cg contained tlic same subst~anccs blit irl diffcrerrt concent,rations: 0.1 filhis-Tris, 0.1 IV-M~~S, 0.05 ~~-2-~nc~rcaptoctl~~~~~ol and 8 wl~rva. Both buffers wrre adjwted to a pH of 6.8. l’lle third l~uf?‘w (d) col~tainerl 0.1 wTris, 0.1 M-t)icille, 0.05 >I-2.mercaptorxtlrarrol atltl 8 ~l-\Irc~a (pH 8.X). Tlrv drtailed c~lccttrol)llor(,tic corlditiotls, t)le staininp wit11 (‘oolnassitl Hrilliarlt Bloc, lt350 and detrsilotrwtr?; of tIl(* dried strips \vas as descritmt by Zubkr ct al. (l977).
Tbis was done on cellulose acetate st,rips as ticwribed by Zltbke et al. (1977) ant1 G. Stiiffler & H. Elrrlich (unpublislled results). E’ollowi tlg cellulose w&ate electroplloresis the strips \vore transferred directly cit,llor into tile wltiselum elicited to R. megaterizcm tIliostreptorr-binding protein or illt)o anti-R. coli LI 1 or into prr-immune sera from t)hcx same animal in control experiments. Excess antisermn was removed by several washes with pllosp~rate-buffered saline and the arttigm antibody rornplt~xes were stained with Coonlassie Brilliant’ Blur R250 (Zubke et al., 1977).
3. Results (a) Binding
qf [35S]thio.str.epto?l to riboaomes. ribosorrral
xubutds
and core
parbides
The preparation of [35Slthiostrepton used hew bound to 50 S ribosomal subunits with 1: 1 stoichiometry (Table 1. lines a and b) hut did not’ bind at all to 30 S subunits (Cundliffe. 1978; see also Sopori & Lengyel. 1972; Highland of ul.. 19751). Accordingly, in the following experiment,s. WC have assumed t’hat whcnevrr binding of thiost’rcpton \vas ohserved it vxs occur-rinp upon 50 S subunits or particles derived therefrom. When a variet’y of IX1 cow particles were produced from 70 S ribosomes of \vild-type B. megateriunz. the; each failed t’o biud [ 3”S]thiostrepton but could bc restored in their ability t,o do so by addin g back 1 M-LiCl split proteins (Table I
240
E. CUNULIF’PE
E’2’ =1L.
TABLE
Binding
1
to ribosomal subunits. core particles und reconstituted derived from ribosomes of R. megaterium wild-t!jpe
of [35S]thiostrepton particles
Ribosomel p8xticles (50 pmol)
a b c
50 s 30 s
THSf houlitl (pmol)
Additions
.~ --
(.I
1 M-Licl 1 II-LiCl
(7oro oorc
e f
2 M-LiCl core 2 M-LXX CON>
#
2 x-IX1
cow
-~. 1 WLiCI split proteins 1 31.LiCl split proteins Purified thiostreptow binding protein (Bhl-LI
1)
i THS, [%]thiostrepton. Reconstitution of LiCl core pLtrt,icles with split prot~eins zrrrti :wsnys of the hio(lrnp ot’ /““Sjthiostrepton were carried out its described in Materials H~CI Methods. Each wsay contoinrd 50 prn(A of core particles or ribosomal subunits which were fini*lly incnbitted with 60 ~nnc~l [ “~S]thi~st,l.(~l)toll (430 cts/min per pmol). Reconstitution involved the ttdditiou of 100 1xnol equivalents of 1 a~.LiC’l split proteins or 100 pmol of purified protein BM-Lll (thiostrepton-binding protein) to 50 pmol of cores.
lines c to f; see also Highland et al.. 19756). Therefore: \I;(: decided t’o purify from 1 nl-LiCl split proteins the protein(s) active in restoring to 2 M-LiCl core particles the ability to bind the drug. (b) Purification
qf a protein
i,nvolved i?~ the bkding
of’ thiostreptot,
to ribosomrx
This was achieved by a two-step procedure involving ion-exchange chromatograph) on carboxymethylcellulose as the first step. The acbivt: component was retained on this column and was eluted by sodium acetate in the concentration range 35 t,o 43 mM (i.e. in the conductivity range 2.1 to 2.3 mmho; see Fig. 1). Active fractions were examined by electrophoresis on SDS/polyacrylamide gels and usually contained three or four proteins with molecular weights of approximately 28,000, 23,000, 17,000 (a minor component when present) and 15,500 (the major component). The second purification step involved chromatography on Sephadex GIOO in the presence of 6 M-UP%. Column fractions were again assayed for their ability to restore thiostreptonbinding activity to 2 M-LiCl cores (data not given). Fract.ions active in this assay contained essentially one protein as judged by electrophoresis on SDS/polyacrylamidc~ gels (Fig. 2(a)), urea/polyacrylamide gels at pH 4.5 (Fig. 2(b)) and polyacrylamide gels run in two dimensions (Fig. 6(d)). Th e molecular weight of this protein was determined to be about 15,500 on SDS/polyacrylamide gels and we estimated tha,t it was at leasb 9S”/h pure from the relative intensities of staining of major a.nd minor bands in these various gels. This single protein restored the ability to hind thiostrepton quantitatively when added to 2 M-Lick core part’icles (Table 1. line g). The minor contaminating component present in this preparation (see Fig. 2(b)) could not be removed and its nature is unknown.
THIOSTREPTON-RESISTAST
RIROSOMES
Fmction
1
241
no.
I“;
28000 23000 15500
f
FIG. 1. Partial purification from B. megaterium ribosomes of a protein involved in thiostrepton binding. 1 M-LicI split proteins from B. megaterkrz wild-type ribosomes were subjected to chromatography on carboxymethylcellulose at pH 5.6 in the presence of urea. Materials retained on the column were eluted with buffer containing a gradient of concentration of sodium acetate. Fract,ions ,)f the eluate were assayed for their ability to restore to 2 M-Lick core particles the ability to bind [%]thiostrepton (see Materials and Methods) and active fractions were analysed by eleotrophoresis on RDS/polyacrylamide gels. -O-O-, Thiostrepton binding; --a-e-, conductivity.
242 Mr markers
24 700
17 000 14 500 13 700
0~1 % SDS/ acrylamide
13% gel
El M-urea/ acrylamide
(a)
(c) Ident%@at%on
IO% gel
(pH4*5)
(b)
qf th,e
thiostrelltolz-Dinding
prolein
Having purified from ribosomes of B. meguterium a protcin \~~hich is principalI> involved in either creating or completing a binding sit,e for t~hiostrepton. n-c sought to identify it by immunological comparison with tjhe riboxomal proteins of E. coli. The thiostrepton-binding prot,ein of B. wzegaterium \las asscxsed by double immunodiffusion with each of the monospecific antisera raised against all 34 proteins from the 50 S subunit’ of E. coli ribosomes. X precipitin hntl WIS formed exclusively with an antiserum to E. coZi ribosomal protein LI 1. none of t hc ot’liw ;lrrtiswa g;rvcl ;w!precipitation (Fig. 3(a) : nega,tive rrsults not shown). Converscl~~. an antiswum raiwcl ‘against the purified R. megateriur~~ protein precipit,ated protcirl I, 1 1 alone and none of the other pure ribosomal proteins fkom the 50 S subunit of E. coli (Fig. 3(b)). These data clearly identif,y the thiostrepton-binding protein of B. megateriuat ribosomes as being homologous to pl,otein Lll of the E. coli ribosome. This pro&in was therefore designated BM-LI 1. anti E. coli Ll I formed a strong prucipitin band In a further series of experiments. with E. coli ribosomal protein Ll 1 but prccipitatrd. altwit, to a Icssrr dcgrw. ribosomal protein B&I-L1 1 of B. ~rrwgatwiuw (Fig. 3(c)). Spur formation Iwt~\wn tlw precipitin
243
I~‘IG. 3. L),,uble irnmunotliffusion. (a) Roaction of protein BM-Lll with antisera against rihosomul pr~kGns from thP 50 8 subunit of E. coli. The ccwtrc will c()ntamcd I pg HM-IAll. The peripheral w-PIIS rontaint : (I) anti-E. coli L7. y-globulin cxres1)ontling to 90 pl wrum-equivalents; (2) ;mtiE. co/i I& y-globulin (300 pl sc~lum-ccluivult~nts); (3) anti-E. coli LS. y-globulin (300 pl srrumequivdcnts) ; (4) anti-E:. coli LIO, 60 pl swum; (,5) snti-E. coli. Lll, 0.2 A,,0 specific antibody (purifktl ovw an h’. coli. LI 1 -:Ainit,y column); (6) arlt’i-E. coli. L12, y-globulin (90 pl serum-equivw lcnts). The ncgat,ivr results for the antisva against the- wmaining 28 ribosomal prokins from t,hc 50 S subunlt of E. coli RW not shown. (b) Reaction of anti-BWLIl with single ribosomal protrins from the 50 X subunit of E. coli. Th(> contw wc~ll contained anti-KM 1, f 1. y-globulin (1750 pl sc~~~~m-c~cluiv;~lent~s). The peripheral ~~11s contnnml (1) 2 pg E. coli. LlO; (2) Z pg E. coZi Lll; (3) 2 p,!q I<. coli LI”: (4) 3 pg E. coli L13; (5) 2 pg E. co&i L14; (6) 3 pg E. coli 1~15; (7) 2 pg E. coli 1‘16; (8) 2 pp E. coli L18. Except for L8, L17 anti LX1 the remaining ribosomal Irotrins from t,hP 50 S subunit of E. COG were tested in t,hc same manner‘ (data not shown).
bands indicat8ed that the proteins are homologous (i.e. scrologica~lly related) but not, identical (Fig. 3(c)). The converse experiment with antiserum raised against, II. WW~Iteriurrl BM-Lll gave a comparable result (Fig. 3(d)). The relatedness of the homologous proteins of the two bacterial strains was further corroborated by modified immunoelectrophoresis. R particularly sensitive technique. Thus (Fig. 4) an antiserum raised against protein BM-Lll formed a single st,rong precipitin band with total rihosomal proteins from B. w~yateriun~ and also with a lone rihosomal protein of E. coli which migrated exactly in the po&ion expected for
244
E.
CUNDLIFFE
E!t’
dL.
43
04
Origin J1
BM-LII
E. coli
L II
FIG. 4. Modified immunoelectrophoresis. 50 pg TP30 from E=. coli (l), 50 pg TP50 from E. coli (2). 25 pg TP70 from B. megaterium (3) and 60 rg TP70 from E. coli (4) were applied t,o the gel. E&&-ophoresis was for 80 min at 360 V and 3.8 mA in buffer d (SW Materials and Met,hods). Strip (b) was soaked in antiserum elicited against, BM-Lll prior to st,aining. Strip (a) was stained directly without soaking in antiserum. TP30, TP50 and TP70 refer to total prot,oins obta,inrd from 30 S rib{)somal subunits, 50 R ribosomal subunits or 70 8 ribosomw. respect~iwly.
protein Lll. It was also clear that protein Lll from E. CA’ and its counterpart (BM-Lll) from B. megaterium migrated differently during electrophoresis which confirms our conclusion that the two proteins are st’ructurally related hut not identical. The converse experiment to the one shown in Figure 4. but employing an antiserum raised against E. coli protein Ll 1. was a,lso performed (data not given) with similar results. We wish to emphasise here t,hat homology b&wren proteins Ll I of’ E. coli and BM-Lll of B. megatarium has been clearly demonstrated using antisera raised against either protein. The antiserum to protein LII was monospecific: and reacted exclusively with pure E. coli ribosomal protein Ll 1 and not with any other ribosomal protein of that organism (SMffler & Wittmann. 1971a.h: Stiiffler. 1974). Comparable experiment)s could not be carried out with anti-RM-Lll since individual ribosomal proteins from B. m,egatrrl;um are not yt+ available. Consequently, t,hc specificity of the antiserum to protein BM-Ll 1 has not’ been as thoroughly demonstrated as that of antiserum bo protein Lll of E. coli. However. the completeagreement between the results obtained with both antisera. described ht:rc, and below! revealed that the antiserum raised against prot)ein BM-LI 1 was also monospecific. The satellite bands visible in Figures 4 and 8 deserve further comment. They wcrt~ formed specifically with antisera raised against protein Lll or BM-Lll and have been seen also with ribosomal proteins from B. subtilis (Wiener1 et ml.. 1979). Since our antisera reacted with a single ribosomal prot,ein (viz. Ll I or its counterpart BM-L11 ) the satellites must, have been serologically related to protein Lll . Apparently, they represented a modified species of protfein Lll (or BM-Ll I ) although whether such modifications were physiological or artefactual remains to hc determined. However. we note in this context that E. c&i protein Lll contains three methylated amino acid
THIOSTREPTON-RESISTANT
RIBOSOMES
245
residues (Dognin & Wittmann-Liebold, 1977) and is the most heavily methylat,ed protein in t’he ribosome (Alix & Hayes, 1974; Chang & Chang, 1975) as is also t#he case for protein BM-Lll in B. megaterium (Cannon & Cundliffe, 1979). It is possible that the satellites represent differently methylated variants of proteins Lll and BM-Lll since differences in the degree of methylation of a protein might reduce. but \rould not be expected to abolish, its antigenicity. In fact, the non-methylated protein Lll from mutant prml of E. coli (Colson & Smith, 1977) was precipitat’ed by an ant’iserum to E. coli protein Lll (G. Stiiffler, unpublished data). (cl) Properties
of’ ribosom,es of thiostrepton-resistant
mutants
of B. megaterium
A number of spontaneously arising mutant strains resistant to t’hiostrepton were obtained from B. megaterium and three of them, designated PDI, PD14 and MJI. are described here. These strains which grow less rapidly t’han wild-type in the absence of drug are tolerant of much higher concentrations of thiostrepton than is the wildtype but are not totally insensitive to thiostrepton. When ribosomes and high-speed supernatant (SIOO) were prepared both from mutants and from wild-type and recombined in reciprocal fashion in cell-free systems synthesising polyphenylalanine, it became clear that the mutants owed their resistance to thiostrepton to some altered property of their ribosomes and that) their post-ribosomal supernatant fractions (SlOO) were not nota,blp different from that of wild-type (data not given). The synthebic activities of rihosomes from mutants PDI and MJI and from wildt’ype were compared in cell-free systems supplemented in each case with wild-type supernatant (SlOO). It was evident that ribosomes from either mutant were only about. 50(;!; as active as wild-type ribosomes even in t’he a’bsence of thiostrepton (Fig. 5). Also. ribosomes from the mutants were significantly more resistant, to the a,ction of thiostrepton in vitro without being tota,lly insensitive to the drug, a pattern which reflects the growth characteristics of these mutants. When ribosomes from mutant NJ1 (Fig. 5(b)) or mutant PDl (d a t a no t given hut similar to that in Fig. 5(b)) UXYY supplemented with protein BM-Ll 1 from wild-type. their synt’hetic activit,ies wew increased to wild-type levels and their resist,ance to thiostrept,on decreased dramat8ically. Similarly, whereas ribosomes from all three mut’ants bound [ 35S]thiostrepton poorly in comparison with t,hose from the wild-type. they did so quantitat’ively when supplemented eit’her wit)h 1 M-l,icl split) proteins from wild-type or with purified prokin BM-Ll 1 (Table 2). (c) Elwtroph)oretic
&di~s
?f rihosomal
proteinn
of thiostrepton,-resistant
mutants
The ribosomal proteins of mutants MJl, PDI and PD14 were compared with t,hostk of \viId-type B. megaterium, by two-dimensional polyacrylamide gel electrophoresis. ‘t’h(~ pa,tt’rrns of spots revealed after staining proteins in the gels were essentially icknt’ical with one not’able difference: the spot corresponding to protein BM-Lll in wild-type (Fig. 6(a) a’nd (d)) was missing from the gels containing ribosomal prot’eins from mutants MJl or PDI (Fig. 6(b) and (c): data for mutant) PD14 not given). Also, the absence of this spot was not accompanied by the appearance of any “exka” spot’ which would have revealed the existence of a mutationally altered protein migrating differently during electrophoresis. The ribosoma,l prot,eins of the three mutants have also been compared with those from wild-type B. megaterium 1, cellulose acetate gel electrophoresis using three different buffer systems (see Materials
240
Wild-type
Plus plus &==-I-@0
Mutant
ribosomes
or minus THS
MJI
ribosomes
BM-LII, I
20 Time
(min)
Ribosomss (50 pmol) WCI‘P incubut,ocl at, 0 ‘C untl at 20 C’ with ~tntl without :I pg of’pr~~tein B&L 11 (see Mat&& anti &lMothods). Sometimes, thiostrepton (:300 pmol) wits then art&xl and incuhat,ir)n continued at 20°C’ for a further 5 min. Synthetic activity of ribosomc>s was then asrayecl at, 37 ‘(’ following addition uf high-sprrd sup~rnatant (5100) from I{. rr~grcteric~,rn wiltl-t~ypcs, t,ogethcr with cocktail oor&aining (l*C ]phonylalanine. polg(U) etc. (WC Mt~t~rials and Methods). Samples (10 ~1) were withdrawn from incubation mixtures, placed in 10~~~ (w/v) t&hloroacetio acid, heatetl at 90 Y’ for 30 min and pr~~ss~~l RX described in Materials ant1 Mlrthotis. Thiost,rept,on (THS) was tlirsolvod in tlimet,hyl rulphoxitk which with ILIW ;trlcIc(l to cvlnt?oi I II cubations at a final concentration of 1.25”i, (v/v). 1 pmol [ 14C]phmyla1anint~ gavr approximtrtc:ly , ribosomcs plus 1000 cts/min under t,hhetir conditions. (- a--(: -, 1Zibosomcrj ;tlorw ; -l,.protein B&I-L11 ; -@----a-, riboaomcs plus thug: --I--& , riboxomt,s l;ius lrotein B&l-1~11 plus drug.
TABLE
Binding Rourne (50
qf 35X lthiostrepton of ribonomrs pmol)
2
to rihosolnrs Atltlitions
of B. megaterium [ “5Nlthioxtrrl)tolr hountl (17m01)
Ribosomea (50 pmol in RS buffer) were incubated for 5 min at O“C then at 20 (’ Lvith t.itht.1 100 pmol of prot,ein BM-Lll from R. megateriune wild-t,ypcx or with 75 pmol equiv&nts of 1 nr-IX1 Rplit proteins from wild-type. Then [93]thiostrepton (60 pmol; 250 cts/min per pmol) was otcltietl and incubation continued at 20°C for a furthor 15 min. The extent of binding of drug to rihosomvs was then assayed by go1 filt8r&ion (see Materials and Methods). f wt, wild-typo.
THIOSTREPTON-RESISTANT
247
RIBOROMES
lb)
Frc:. 6. Analysis by 2.dimensional polyacrylamide gel electrophoresis of tot,al ribosomal prokin* from 11. megnterium wild-t,ype and mutants, and of purified protein BM-Lll. The gel system was based upon that of Kaltschmidt & Wittmann (1970) as described in Mat~crial~ and Methods. Elecfrophoresis in the 1st dimension (pH 8.5, 4% ( w ./ v ) acrylamide) was curried out at 4°C for 12 h at 300 V anode-to-cathode for basic proteins and for 4 h at 150 V (also at 4°C’) cathode-k-anode for acidic proteins. Electrophoresis in the 2nd dimension (pH 4.5, 18% (w/v) acrylamide) was carried out at 4°C for 20 h at 120 V anode-to-cathode. Proteins in the final slab gels were stained with Coomassie Brilliant Blue G250 (0.04?4 (w/v) in 3.5% (w/v) perchloric aciti) and were destained using a mixture of 15% ( v / v ) mrt,hanol and 7.5% (v/v) glacial acetic acid. So differences were detected between the acidic ribosomal proteins of the wild-type and any mutants and, for ease of presentation, data relat,iug to acidic proteins are not given in Pig. 6. (a) Basic ribosomal proteins from N. megntwium wild-type; (b) basic ribosomal proteins front mutant MJl ; (c) basic ribosomal proteins from mutant I’D1 ; (d) purified prot,oin BML11 (thiostr~:l,ton-~,intling protein) from wild-type.
and Methods). It was found that a single protein-band st’rain. was absent in each of the three mutants (data The dat*a presented in Table 2 and in Figures 5 and 6 from mutants MJl, PDl and PD14 either lacked protein counterparts of BM-1~11 which co-electrophoresed with from which they could not be distinguished on standard resolved by examining the ribosomal proteins of the w&h wild-type by immunological techniques.
which is present in t’he parent not shown). clearly indicate that ribosomes BM-Lll or possessed variant, some other ribosomal prot,ein(s) gels. These possibilities were mutants and comparing them
E.
248
(f) Immunologicnl
properties
CUNDLIFFE
ET
of rihosomal
A I,
pofei1r.v
of ihiostrrl,torl-rp.si.st(~llt
rnutatrt.s
Total ribosomal proteins from wild-t,vpr, H. r//e@eriurrr aud from muta,nts t’l)l. PD14 and MJl were examined by immunodiffusion with antiserum raised against ribosomal protein Lll of E. COG (Fig. 7(a)). Proteins from the wild-type produced w weak precipitin band whereas those from the mutants did not. These initial results supported two contentions: namely, tha.t the protein altered or absent in the mutants (as compared with wild-type) is related to E. COG protein Lll and t,hat ribosomes of
(a)
(b)
FIG. 7. Double irnmunodiffusion oxlxximent~a with (a) The centre wall cont,ainod 250 pl anti-E. coli Lll. TP70 from H. meycslerium wild-type; (2) 60 pg and (3) PDl; (4) and (5) blank; (6) 100 pg TP70 from IY. eoli: (b) The centre well contained 260 pl anti- /I. megrcteriurn TP70 from B. meycrterium wild-t,ype.
(1) 100 pg TP70;
(2) 150 pg TP70;
ribosomal lw&eins from mutant 1’l)l. Tho peripheral wells wnt,aincd: (1) 60 pg IN pg TP70 from tl. rnogtrteriz~m mutjant (7) and (8) bla,nk. HM-LI 1. Thv priphwal w~~lls rontarnt~l
(3) 200 pg Tl’iO.
the mutants do not contain any protein able to be precipitated by t,he ant,iserum raised against protein Lll of E. coli. This conclusion was further substantiated using the antiserum against protein BM-Lll which reacted more strongly with the homalogous antigen in ribosomes of B. megateriumn wild-type and could therefore give morci rigorous evidence as to whether or not the ribosomes of the mutant,s contained protein BM-Lll. Again, ribosomal proteins from the wild-type but not Ohose from the mutantti formed precipit’in bands with this antiserum (Fig. 7(b) and (c)). This result. strongly suggests that ribosomes from the mutants do not contain ribosomal protein BM-I,11 . However, since negative immunochemical results cannot always bc regarded as conclusive. we attempted to confirm these result’s using a, more sensitive technique. namely modified immunoelectrophoresis (Fig. 8). This method is not simply based on immunoprecipitation but also on binding of specific antibody to the antigen which is immobilized on a cellulose acetate gel (Zubke ~1 al., 1977 ; G. Stiiffler t R. Ehrlich. unpublished results). This technique has proved to be about an order of magnitudtx more sensitive than Ouchterlony double immunodiffusion in det,ecting strong antigens and has even allowed the detection of certain weaker antigens (e.g. fragments of ribosomal proteins) which do not form precipitin bands with antiserum during double immunodiffusion (R,. Ehrlictr & G. Stiifflcr, unpublishad data). Again (see also
THI08TREPTOS.RESISTrZXT
249
RTBOSOMES
e
oe
Origin J
f BM-LII 25 pg (1 anti 26 pg (4), 37.5 Elcrtmphorcsis Prior to staining
7) ad 12.5 pg (2 anti 8) of TP70 pg (5) and 50 pg (6) of TP70 from was for 100 min at 250 V and 3.7 the strip was soaktd in antiswum
of
11. megtctarium wild-type; and 12.5 pg (3) II. megrrterium 1’111 were applied to the gel mA4 in buffer e, (SW Materials and Methods) clicitetl against B&l-Ll 1.
Fig. 4(b)) a strong precipitin band plus a weaker sa,tellit’e was obtained with B. megateriurn wild-type ribosomal proteins in response to antiserum specific for protein BM-Lll. In contrast,, no precipitin ba,nds were obtained wit’h ribosomal proteins from any of t#he three mut’ants even when a’ broad range of antigen concentrations was assessed. We therefore concluded that we had eliminated all but t,he remotest possibility that any cross-reacting material related to protein BM-Lll was present on t*he ril)osomes of mutants MJl, PDl and PD14.
4. Discussion Ribosomes of Bacillus megaterium contain a protein (BM-Lll) which is immunologically related to protein Lll of E. coZi ribosomes. These two proteins have similar molecular weights, namely 15,500 as estimated for protein BM-Lll (Fig. 2(a)) and 15,300 as estimated for protein Ll 1 by a similar technique (Zimmermann t Stoffler. 1976) and are further related by their ability to create or complete a ribosomal although neither protein binds thiost#repton off the binding site for thiostrepton, ribosome. Proteins Lll of E. coli and BM-Lll of B. megaterium are also related in that they are the most heavily meth,vlat,ed prot’eins in their respective ribosomcs (see above). Although no biological role yet postulated for protein Lll has been related to the fact that it is methylated, the protein itself has been implicated in various aspects of ribosome assembly and function. Thus, protein Lll binds to 23 S RNA in, vitro under condit’ions where t,he protein is not denatured (Lit~tleahild rot al.? 1977) and is
260
E.
CUNI~LIFFF,
ET
A L.
involved in integrating protein Ll6 into t,hc peptidyl t,ransferaw centjrc oft tw ribosonw (Diet)rich it ccl., 1974; Moore et al.. 1975). ItI has also been sugg&ed t,hat protrin I,1 1 is crucial for the expression of peptidpltralisf~rase ac!t,ivit?, (Nierhauw & ?dOtlt~fb,~fJ. 19%‘) but, this conclusion is hardly compat’ibk, \vit,tt our data. since l~ihMCJlllC~S ot mutants PDl, PD14 and MJl which lack protein BM-LlI are act,ivr in prot,t+n synthesis i7~ vitro (see Fig. 5). Other investigators have also shown that, protttin I,11 is not obligatory for peptide bond formation (Howard & Gordon, 1974: Ballesta $ Vazquez, 1974: Moore et al.. 1975 ; Armstrong & Tate. 1978). Prot,ein Lll is lab&d by a photoactivated analogue of GDP in the presence of elongation fa,ctor EF-G (Maasen & Miiller, 1974) and has even been postulated to be t)he ribosomal GTPaw enzyme (Schrier & Miiller, 1975). Cross-links have also been formed between pr&oin Lll and proteins L7, L12 and L10 (Expert-Bezanqm rf al.. 1976) and also proteins L2, L4 and L14 (Kenny & Traut, 1979). These proteins arc in the neighbourhood of the ribosomal binding domains of the protein synthesis fact,ors IF-2, EP-Tn. EF-G. RF-1 and RF-2 and are close to the site where GTP hydrolysis occurs (for reviews see Miiller, 1974; Miller & Weissbach: 1977: Brat,, 1977). Mutants of E. coZ?Iwith variants of protein Ll I have been isolated and have in some cases (reZC) been shown t’o exhibit relaxed control of RSA xynt’hwis (P>lrk(lt of al., 1976). Sinor thiostrepton also inhibits thrl ri~)osonlc,-tlcp(,I~~~~,rlt formatiotl of’ guanosinc t,c+a- and penta-phosphates in extra& of stjringc~ntj (~11s (Hawltiw A r/l.. 1972 ; Pederscn rf al.. 1973) in addition t)o exerting t htx other c+Yccts outlitwtl t~at~lit~r. thcrc is a substantial correlation between post,ulated functions of prot,cLiu Ll I antt processes inhibited I)y thiostrcpt,on. Howevrr. in viw of our obs~rv;~tion t tlat mutants MJl. PII1 and PD14 possess func~tional ribosomcs \vhich 1ac.k ~~rotckr BM-Lll, no indispensable function can be attributcld to this protein (or t)y inti~t~c~tlc~c~ to protein Lll of E. coli). This swvcs to tmphasiw thr itnportaticc of cw-c)pcwti\-ts interactions bet,\veen ribosomal components in thrl cxprcssioti of their functions. Our dr>monstration t,hat ribosomes frotn muta&s M,Jl ant1 I’DI (resistant to thiostreptjon) are restored to lvild-type levels of act,ivity and drug scansit ivit y \\.trrw supplemented wit,11 prot’cin BM-Ll 1 might have bwn caxplioa 1)1(xin terms of r~xc+hatryc~ of proteins between the ribosomc-bound and free forms. particularly sitrw proteitt Ll 1 of E. cnli has been shown to undergo such cxchangc ire ,vitw (Rohrrtson c/ (41.I 1975: Subramanian & van Duin. 1977). WC therefort: felt ii- (~ncutnlwnt upotl 11s to establish b!- t,he most, rigorous means possible that rilwsotrrcs ftwtl out’ tnutatlt s atv deficient in protein HIV-TA 1. It is quite difficult to establish unambiguously \\~hethcr ot’ not’ a protcliu is missing from ribosomes. The difficulties in reaching such a conclusion in the (:atit’ of wrcral E. coli mutants which possibly lack protein 820 ww t,xtcnsi\-oly discuswd 1)~. Witjttnann nf al. (1975). For t’his reason we exatnined t)hc\ ri hosotnal prot’c+ns of out’ mutants I)y WVtbrill c~lcctrophorot,ic and irnmutloohc~mici~l tI&niques. Ttith twults obtainctl wtw in complctc accord and indicated t’hat prot,cin BM-I,1 1 is missing from the ribosomcs of our thiostrept,on-resistant mut,atits. t n particular. wheti ttlodificti immutioclt~ct~ropt~oresis \vas employed for a rigorous c~xwminatioti of the t~ihS~~I~li~l proteins of mutants PDl. PD14 and MJl~ no cross-reacting tnatt~tial rc~latetl to eit,hrr H. megatprium protein B&I-L1 1 or E. coli protein LI 1 \vas detected. W’V COIIsider this data as being most convincing. Accordingly, wc conoludo that) no countwp;l rl of prot’ein BM- Ll 1 whether drastically altered or undt~rmet~1yI;lt~ecl or evcm an ‘.a trtltc~t~” fragment,, \\‘as present, on the rihosomcs of the t trrtw mut,ants studied.
‘I’HIORTHF:I’TON-IZE:SIHTANT
RIHOSOhIES
2.5 I
We then sought to establish whether cross-reacting material related to protein BN-IAl was present elsewhere in our mutants. Conceivably such material might not ha,vcx INXI~ assembled into ribosomes of the mutants or might have been lost from the ribosomes during their preparation. The latter case is perhaps unlikely since residual traces of protein BM-Lll should have been detected on t,he ribosomes. Preliminar) titudies revealed such cross-reacting material in acetic acid extracts from total cells a,nd in the SlOO fraction. Analysis by double immunodiffusion revealed only pnrt’ial serological identity between protein BM-Lll and the cross-reacting material as judged from the formation of a spur. We are unsure of the nature of this cross-react,ing material and further studies will be needed to characterise it. However, our abilit’y to detect such cross-reacting material among the total cellular proteins of the mutants proves that t,he modification(s) introduced by mutation did not prevent prc:cipitation of some form of protein BM-Lll bg the antiserum. This reinforces our conclusion that’ had a,ng such material been present on the ribosomes of the mut’ants we should have detected it. Results similar t,o some of those report,ed here have also been obtained indepcndent]?; and concurrently by others. Thus, certain mutants of B. suOtiZis(Goldthwaite & Smith, 197%; Pestka et al., 1976: Wienen et nZ., 1979), arising spontaneously and resist’ant to thiostrepton, possessed properties somewhat similar to those of mutants I’DI. PI)11 and MJl. Furthermore, wild-type B. mbtilis cont’ains a ribosomal protein ES-I,11 \j.hich is relat,ed to E. coli protein Lll and to BIN-11 bot,h immunologicall! ittld in its allility to promote the binding of thiostrepton to ribosomal core particles (N?clnen et al., 1979). Six t’hiostrepton-resistant mutants lack protein BS-Lll ; the protcitl ~~1s found t,o recur in six spontaneously-derived rev&ants (Wienen et al.. 1979). ‘I’llis \vork TVASsupport)ed by grants from t,he Deutsclle E’orsclturl#s~es~~llscllaf; (to U.S.) at~d t Ilrs Medica. Research Council (t,o E.C.). We are also indebtetl t,o MRC for Researclt Strlctcwtslrips (for P.D. and M.S.) and to Professor H. G. Wittmanrr and Dr M. J. Dognit for pro\-iding puw E. coli rihosoma,l proteins. The patient criticism of E. R. Dabh ant1 B. \\‘ic~netl is gratefully acknowledged. I)r C’. (‘olson kindly provided rnlltantj ,urml of E. co/i.
R EI’FREYCES i / .\lis, .I. H. Jt Hayes, D. (1!)74). ,I. 3Iol. Riol. 86, 33% 150. Armstrong, I. L. & Tate, IV. P. (1978). J. Blol. Biol. 120. I55 166. Ballcsta. .J. P. G. & Vazqnrz, D. (1974). FEBS Letters. 48, 266 270. Brat, S. (lRi7). In AIoZecular Mech,anisms of Protein Biosyn.thesis ( Weissbacl I, H cY: I’wtka. 8. cds), pp. 375-411, Academic Press, New York. CJatrrlorr, M. 8z Cundliffe, E. (1979). Eur. ,I. Biochem. irl t)he press. (Ilmtly, 1’. I\;. B Cllang, C. N. (1975). Biochemistry, 14, 468-477. Cohn, (‘. & Smith, H. 0. (1977). Afo/. Gen. G’enet. 154. 167 173. Clutdliffr. E. (1971). Biochem. Biophys. Hes. Commun. 44, 912 917. CrulcUiffe. E. (1978). Nature (London), 272, 79% 795. (Irtl~dliffe, E:. (1!)79). III Antibiotics (Hahn, V. E., (~1.). WI. 5, pp. 329 343, Sprill$z(‘t.Vrrlag. NC,\\, YC ,rk Dirtricbll. S.. Sclwarrdt~, I. & Nirrllatls, K. H. (1974). Jf’EtiS Letters, 47, I36 139. Dixon. I’. D., Brvrn, ,J. E. & Cundliffc, E. (1975). ilntimicrob. dg. Chemother. 7, 85Ob855. Dognitl. M. .J. & Wittmann-Lieloold, R. (1977). &‘EBS Letters. 84, 342 346. l~~xl)‘rt-Rcz;trlsorl. A.. Harritault, D., Millet, M. 8: Hayes. D. H. (1976). ,J. Mol. Biol. 108, 781 787. (:c)ldtlr\vnitl~, C’. & Smitll, 1. (1972). ~~Iol. Cen. Oenet. 114, I!)() 204.
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b,-f/’ :lL.
Haseltine, W. A.. Block, R., Gilbert, \V. & Wrbrr. K. (1!)7%). LYat?rre ( /Jontlorr), 238, 381-384. Highland, J. H., Howard, U. A. & Gordon, .J. ( 197Ba). &w. ./. H%ochem. 53, 313 :+I 8. Highland, J. H., Howard, C. A., Ochsner, E., Stiiffler, G., Hawtrbank. Ii. & Gort1011. .I. (1975b). J. Biol. Chem. 250, 1141-~1145. Hindermach, I.. Kalt,schmidt, E. & Wittmann. H. (:. (1971). E~rr. ./. Hiocherrr. 23. 12 Iti. Howard, (:. A. & Gordon. J. (1974). li’ERi3 Letters, 48, 271 27-C. Howard, G. A. & Tmut,. K. H. (1973). FEBS Letters. 29, 175 180. Kalt,sctnnidt,, E. & Wittmaun, H. C:. (lU70). AT&. Riochem. 36. 401 412. Kenny, J. W. & Treut, H,. R. (1979). .J. Mol. Bid. 127, 24% 263. Laommli. U. K. (1970). &‘&we (London), 227, 680 685. Lieu, Y-F., Yoshikawa. M. & Tanaka. N. (1975). Hinchem. Hiopl~~ys. 12rs. C’ornm~~~~, 65. 1096 1101. Littlrchild. ,I . . Uijk. .J. & Garret,t, H. 4. (1977). I’8IHA’ /,etters. 74. 29L d!fl. Maassen, .J. A. & Mailer. W. (1974). I’roc. ,\“at. AC&. A’ci., l:.S.d. 71, 1277 1280. Miller, D. I,. & Wnissbach. H. (1977). In Molecular Mechaw&ms of J’rotein Biosgr~thesis (Woissbach, H. &, Pcst,ka, S. eds), pp. 323 373. Academic Press, Nwv York. Modolell, .J., (‘abrw. Is.. Parmrggialli. il. K- 1’azq~~~:z. I). (1971). Z’ror. ,\Tat. .4catl. ,Sci.. l/.S.d. 68. 1796 1800. MGller, W. (1974). In Rihosomes (Nomura. M.. TissiBrw. A. k Lr~rrgycl. I’. eds), pp. 71 1 731, Cold Spring Harbor Laboratory, New York. Moore. V. ct.. Atcllison, R. E., Thomas. G.. Morall. M. &, Nollcr. H. P. ( 1975). I’Tw. LYaf. Acatl. ~Sci., TT.S.A. 72, 844-848. Nierhaus, K. H. &. Mont,rjo, ‘I’. (1973). I ‘roc. ‘Vat. .-Icat/. Sci.. [‘.X,.-l. 70. I931 1935. Ouchterlony. ii. (I 968). Ha&hook of immunod@&o?l an,n’ i,nmlcrcoelect~o~hore.Fi.s, .AIH~ Arbor Scier~w E’ublishers. ,Antl Arbor. Michigan. Parker, ,I ., Watsotl. K. ,J., J+irserl. ,J. D. $ Fiil. N. P. (1976). XO/. Gan. (ienet. 144, I 1 I I I-&. Prderserr, F. S.. Lund, H. $ Kjrldpaard, Iv. 0. (1973). Nature (I,ondovc), 243, 13 15. Pestka.. S.. \Veiss, II.. Vitwr. R.. Wic~rwtl. IS.. Stiifflw. (4. $ Smith. 1. (1976). A/al. (;cI~. Nenet. 144. I.‘)‘!I’,1 241. Robertson, M’. K... l)owset,t, S. .J. & Hardy, H. A. S. (1!)77). Mol. (:er,. Cienet. 157. ‘05 214. Schrier. I’. I. & Miiller, W. (1975). FE&S’ Letters. 54, 130- 134. Sopori. M. I,. & Lmgyvl. P. (1972). Niochem. Bioph,,ys. Res. C!omvnctr/. 46. 238 244. StGffler. (:. (1967). ;210/. Get,. f:enet. 100, 374 377. Stiifflw. (:. (1 !)iJ;). 111 /
(1979) 132, 235252
Ribosomes in Thiostrepton-resistant Mutants of Bacillus megaterium Lacking a Single 50 S Subunit Protein ERIC CUNDLIFFE, PETER DIXON, MICHAEL STARK Department
of Biochemistry, University Leicester, England
of Leicester
GEORGST~FFLER, RENATE EHRLICH,MARINA ST~FFLER-MEILICKE Max-Plan&-Institut
fiir Molek,ulare Berlin-Dahlem,
Gelzetik, Ahteilung
Wittm,ann
Germany
AND MICHAELCANNON Department of Biochemistry King’s College, University of London London, England (Received 28 December 1978, and in revised ,form 11 April
197’9)
A protein required for the binding of thiostrepton to ribosomes of Bacillus megaterium has been purified and furt,her characterized by immunological techniques. This protein, which does not bind the drug off tllc ribosome, is serologically-homologous to Escherichia coli ribosomal protein Ll 1 and is designated BM-Lll. Ribosomes from certain thiostrepton-resistant mutants of R. megaterium appear to be totally devoid of protein BM-Lll as judged by modified immunoelectrophoresis. Such ribosomes are significantly less sensitive than those> from wild-type organisms to the action of thiostrepton in vitro but retain subst,ant,ial protein synthetic activity. Re-addition of protein BM-Lll to ribosomrs from the mutants restores them to wild-type levels of activity and thiostrepton sensitivity. Thus ribosomal protein BM-Lll is involved not only in binding thiostrepton but also in determining tile thiostrepton phenotype.
1. Introduction binds to bacterial ribosomes and prevents their functional interaction with various protein factors involved in the initiation, elongation or termination stages of polypeptide synthesis (for a review see Cundliffe, 1979). Consequently. hydrolysis of GTP, dependent upon the binding of such factors t’o ribosomes, is inhibited in vitro. The observation that the closely related antibiotics thiostrepton and siomycin are able to inhibit functions involving both the elongation factors EF-Tu and EF-G led to the idea that ribosomes possess a single site which is utilised by these factors alternately and which, together with either factor, const,itut’es a GTPase centre (Cundliffe, 1971: Modolell et al., 1971). Rihosomes of Escherichia coli possess a single binding site for thiostrepton, located 235 4‘ 1979Alcadenlic Prow Inc. (I~~nrlon) Ltcl. 00”“~ 283A/79/%20235-18 $02.00/o
Thiostrepton
236
E. C’~TN1~I,Il~FE
ET
AL.
on the 50 8 ribosomal subunit (Sopori & Lengyel. 1972: Highland it nd.. 1975~1,).tc, which the drug binds with high affinity. This site is disrupted why 50 S subunits it1.t’ exposed to 1 M-LiCI, thereby producing “I M-LiCI (~JI’O partjicl~~s”. I)ut (aan 1~. I’(‘rt (I/.. 197%). \ic’hct~ assembled IJ~ adding back ribosomal prot,ein IA I (Highlantl 1 M-Lick split proteins were treated with ant&ra raised against various purified ribosomal proteins, only immunoglobulin raised against protein I,1 1 abolishrtl thth ability of split proteins to restore to cores the abilit,y to hind thiostrepton. Xccordingly and as a result, of other experiments, it was concluded (Highland it ~1.. 197,%) that protein Lil is required for the binding of thiostrepton to riboxomes of E. co/i. Experiments involving the characterisation of antibiot,ic- binding sites by partial reconstitubion of ribosomes from core particles a,nd split proteins can br usefUy complement’ed by examination of mutationally altered riboaomex ohtaincd f’rom drug-resistant mutants and also by binding studies with aflinity analogurs of antibiotics. In t,his work we did not use t,hiostreptoll-I,esist,ant mutants of KF:.loli sinc*cs the wild-type of that, organism does not take up the drug. Mut,ants of E. c,o/i \vhich allow penetration of thiostrepton can only h otjtaintd hy tlrcs ww of’ tnutagws (Liou et ul., 1975). a procedure \vhich we wished to avoid. Xocordingly. w(& Ilavt* examined the ribosomal binding s&e for thiostrepton ill Racdllus /r,egaf~~irrnr ill1 organism which is sensitive to the drug. Here wt’ report that ribosumes of B. tttegrcterium contain a protein which is immunologically relat)cd to ribosomal protoitl LI I of E. ~4% and which is involved in the binding of thiostrepton to ribosomrs. 1n addition. we discuss the properties of the ribosomes of three spontnnr~ousl~~ arising mutant strains of B. vtz~egaterium which are resistant8 to tjhiostrrpton.
2. Materials and Methods (a) Selection
of mutart&
and maintena?rce
of strains
Mutants of 13. megateriwm KM we’re selected by plating the wild-typo on Irutrient :rgur containing thiostrepton and arose spontaneously with a frequrncy of approx. 1 ill 10y. The mutant designated MJl was selected on 3 pg thiostrepton per ml of nutrietlt agar whereas mutants PDl and PD14 were selected at 10 p*fs drug per ml. Mutants werct maintained on nutrient agar plates cont,aining 3 pg thiostrept,on per ml, tha minimull growth-inhibitory concentration for the wild-type under these conditions being appros. 0.1 pg drug per ml. In the absence of drug, all the mutants studied CIWC:gram \vith a nleat~ gelleratiotl tjltrrcL approx. 2.5 times great,er than that of the wild-t.ype. Accordingly, when growing t,lrv mutants in bulk, starter cultures cont,ained t,hiostrrpton although growtll was necessaril), continued in the absence of drug. Subsequent, examination revealed that thp rrrlttatltjs were quite stable in the absence of drug and that revertams to wild-type did not arise during bulk growth of mut)ant,s. (b)
Preparation
of ribosomal
core particles
ad
s&t
protein~s
Cells were broken by passing them t,wice t’hrough a chilled Frencll Press operated a~ approx. 12,000 lb/in2. During tllis procedure the concentzation of cell-paste was approx. 500 mg wet weight, of cells per ml in buffer containing 10 m&l-Tris-acet,ata (pH 7.6 at 2O”C), 10 mlvr-magnesium acetate, 50 mM-ammonium acetate. 3 mM-8-mercaptoet~ianol. 0.5 mM-EDTA. Deoxyribonuclease (5 pg/ml) was then added and after 5 min at 0°C: cell debris was removed by centrifugation at 30,000 g for 30 min. Ribosomes were their prt*cipitated by centrifugation in the Beckman Ti70 rotor at 50,000 revs/Knin for 6 11 and the, post-ribosomal supernatant (5100) was dialysedat) 0°C against, 3 x 100 vol. of ribosomc~ storayts storagca buffer (RS bluffer) buffer before being stored a,s samples at - i’O”(‘. Ribosnme
THIOSTREPTON-RESISTANT
RIBOSOMES
237
cont,ained 10 m&I-Tris.HCl (pH 7.6), 10 mM-MgCl,, 50 mM-NH&l, 3 mm-/3-mercaptoethanol. Crude ribosomes were resuspended in RS buffer and were washed once by layering thorn over RS buffer containing 40% ( w / v ) sucrose and centrifuging them for a minimum of 7 II at 50,000 revs/min in the Beckman Ti70 rotor. The resultant “sucrose-washed” ribosomes were resuspended in buffer containing 10 mM-Tris.HCl (pH 7*6), 30 mM-MgCl,, 1 &I-NH,Cl, 3 mM-P-mercaptoethanol and were layered over similar buffer containing 20:/, (w/v) sucrose. They were then centrifuged at, 50,000 revs/min for 5 h in the Beckman Ti70 rotor. This “high salt” washing procedure was carried out 3 times before ribosomos were resuspended in, and dialysed against, RS buffer prior to storage as samples at - 70°C’. Core particles were produced in buffer containing 10 m&I-Tris*HCl (pH 7.6), 1 rn>IMgCl,, 3 mM-8.mercaptoethanol together with LiCl (1 M, or 2 M), to which ribosomes were added at 2 mg/ml final concentration. After 17 h at O”C, core particles were collected by centrifugation at 45,000 revs/min for 6 h in the Beckman Ti75 rotor and were resuspended in, dialysed against, and stored at -70°C in, RS buffer. The LiCl extract which contained split proteins was concentrated by dialysis against RS buffer containing 207; (w/v) polyethylene glycol (PEG 6000) at 0°C. Following low-speed centrifugation, of approximately split proteins were st,ored in RS buffer at - 70°C at concentrations 4 nmol equivalents per ml. One nmol equivalent, of split proteins was defined as the amount obtjained from 1 nmol of ribosomes. (c) Assay for bindiny
of [35S]thiostre@on
to ribosomal
particles
to This was carried out as previously described (Dixon et al., 1975) in buffer identical that used for storage of ribosomes, cores and split proteins. Cores or ribosomes, usually 25 t,o 50 pmol in 50 ~1 RS buffer were mixed with 5 ~1 [35S]thiostrepton (60 pmol) dissolved in dimethylsulphoxide. After 10 min at room temperature 100 ~1 buffer was added and the whole incubation mixture applied to a column of Sepharose 6B or Bio-Gel Al.5 m (5 cm x 0.55 cm) equilibrated with RS buffer. Binding was determined by estimation of radioactivity in tile void volume by liquid-scintillation spect’rometry. Various preparations of [35S]tlliostrepton were used during the course of these experiments at specific radionativities in the range of 200 to 500 cts/miri per pmol. (d) Reconstitution
of ribosomal
particles
from
cores and split
proteins
This was carried out according to Highland et al. (19753). Cores were mixed with 1.5 to 2 excess equivalents of split proteins and kept at 0°C for 5 min and then at room tempc,ratllrc for a further 5 min. Radioactive thiostrepton was then added and its binding assayed as above. RS buffer was used throughout. (e) Chromatography
Split
of split
proteins
on carboxymethylcellulose
proteins
were obtained from 900 mg of NH,Cl-washed ribosomes of wild-type B. meyaterirur~ KM by extraction with I M-Lick. These “1 M-LiCl split proteins” were dialysed into 2qb (v/v) acet,ic acid and concent,rated to 20 ml final volume by dialysis against 276 acetic acid containing 20% (w/v) PEG 6000. Concentrated split proteins were then dialysed exllaustively against CM buffer (6 Al-urea, 30 rnM-methylamine, 1 m&Idithiothreitol, pH 5.6 with acetic acid). The 1 M-LiCl split proteins in CM bufl’er were loaded at room temperature onto a column (14 cm x 1 cm; bed vol. 11 ml) of carboxymethylcellulose (Whatman CM-23) equilibrated with CM buffer. The column was washed with 10 ml CM buffer and then developed using 100 ml of a 0 to 100 InM gradient of sodium acetate in CM buffer. Fractions (1 ml each) were collected and samples of each were analysed by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and were assayed for their ability to restore to 2 M-LiCl core particles the ability to bind [35S]thiostrepton. “Active fractions” were pooled (see Fig. 1). They were then desalted over Sephadex G25 (equilibrated with O.lOi, (v/v) acet,ic acid), lyophilized and re-dissolved in 1 ml CM buffer. (f) Chromatography
on Sephadex
GlOO
Following ion-exchange chromatography on carboxymethylcellulose, proteins were further fractionated on a column (100 cm x 1.5 cm) of Sephadex ($100 in CM buffer.
‘I’HIORTICEl’TON-RESIS’~.~~NT (rn) lmm~tnochemical
,methot/s
Atlt~isera to E. cull: ribosomal protein Lll were prepared (Stiiffler. 1974; St,iiffler 85 Wittmann, 197la,b). (II)
I’reparation
of antiserum
“:l!f
RIHOSOMl3S
to the H. Ineqterillrn
a~ld characterized
thiostrepton
as describvd
-hl:ndinq proteirl
A slletp (H80) was irnmutGzet1 \vitll a total alnount of 70 1~ of the protein, purified from R. m,eqaterium 70 S rihoxotnes. Tlrr prc&ein was dissolved in 3.5 ml of 8 M-urea alld stored a.t -40°C in 7 samples of 0.5 ml cacb. Prior to immunization each sample was diluted wit’h 2.0 ml of pllospllatc-bllffrrcd saline (PBS) anti t’he solution emulsified with 2.5 ml i+‘rctlrrtl’s complete adjlivant (lXfc0). Irrjcrtions were administered intramuscularl> atld s~lbcntatieo~lsly at 8 different, sites on days 0, !I. 15, 18. 22, 56 and 60; the animal n-a.s bled on days 0, 27. 33 and 64. Tlrr alttisera \vew incallbatcd at 56°C for 30 mitr to inactivate cotnplrmer~t. For some exprrimcnt~s irrnn~~no~l~~hnlills XVCI’(’ oorlwtlt~ratctl by precipitatiolr a1 40”~,, satrlratioll \vitll ammonitnn sldpllntv. Tlrv precipitates \+c>I’P dissolved it1 L’ES. dialysetl ansinst tllc* same brlfler alld stored at - 40°C’ or lyoplrilized and st)ored a,t 4 (‘. Dvtaiis of tlrr> spwificity of tlris arltiserllm ark prewtltc~
Experirncnts \vere performed (Olvzhterlorlv, 1RO8), with modifications, as tlecnssa~ry for tllc arlalysis of ribosomal protrirrs (Stiiffler & \Vittmanll. 1Wla.h: St)iiffler, 1974).
Tllis was according to Stiiffler (1967) u it h millor modificatiorrs (Zubke et al., 1977; Wierwrr et al., 1979; G. Stijfflrr & K. Ehrlicl~. unpublished dnt,a). Thrco buffers were used: bttffer ,>1 contained 0.12 nz-bls-Tris ([t~is-(2-lrydrox~~~tl~~~l)irrrir~o-tris-(l~ytlroxymetl-~yl)mctlrane]), 0.12 N-MN (2-1~-lnorplrolirro ctllatrv sr~lpl~on~c acid). 0.05~1.2.~nercaptoetllanolitlld 8 ai-nrea. Buffer cg contained tlic same subst~anccs blit irl diffcrerrt concent,rations: 0.1 filhis-Tris, 0.1 IV-M~~S, 0.05 ~~-2-~nc~rcaptoctl~~~~~ol and 8 wl~rva. Both buffers wrre adjwted to a pH of 6.8. l’lle third l~uf?‘w (d) col~tainerl 0.1 wTris, 0.1 M-t)icille, 0.05 >I-2.mercaptorxtlrarrol atltl 8 ~l-\Irc~a (pH 8.X). Tlrv drtailed c~lccttrol)llor(,tic corlditiotls, t)le staininp wit11 (‘oolnassitl Hrilliarlt Bloc, lt350 and detrsilotrwtr?; of tIl(* dried strips \vas as descritmt by Zubkr ct al. (l977).
Tbis was done on cellulose acetate st,rips as ticwribed by Zltbke et al. (1977) ant1 G. Stiiffler & H. Elrrlich (unpublislled results). E’ollowi tlg cellulose w&ate electroplloresis the strips \vore transferred directly cit,llor into tile wltiselum elicited to R. megaterizcm tIliostreptorr-binding protein or illt)o anti-R. coli LI 1 or into prr-immune sera from t)hcx same animal in control experiments. Excess antisermn was removed by several washes with pllosp~rate-buffered saline and the arttigm antibody rornplt~xes were stained with Coonlassie Brilliant’ Blur R250 (Zubke et al., 1977).
3. Results (a) Binding
qf [35S]thio.str.epto?l to riboaomes. ribosorrral
xubutds
and core
parbides
The preparation of [35Slthiostrepton used hew bound to 50 S ribosomal subunits with 1: 1 stoichiometry (Table 1. lines a and b) hut did not’ bind at all to 30 S subunits (Cundliffe. 1978; see also Sopori & Lengyel. 1972; Highland of ul.. 19751). Accordingly, in the following experiment,s. WC have assumed t’hat whcnevrr binding of thiost’rcpton \vas ohserved it vxs occur-rinp upon 50 S subunits or particles derived therefrom. When a variet’y of IX1 cow particles were produced from 70 S ribosomes of \vild-type B. megateriunz. the; each failed t’o biud [ 3”S]thiostrepton but could bc restored in their ability t,o do so by addin g back 1 M-LiCl split proteins (Table I
240
E. CUNULIF’PE
E’2’ =1L.
TABLE
Binding
1
to ribosomal subunits. core particles und reconstituted derived from ribosomes of R. megaterium wild-t!jpe
of [35S]thiostrepton particles
Ribosomel p8xticles (50 pmol)
a b c
50 s 30 s
THSf houlitl (pmol)
Additions
.~ --
(.I
1 M-Licl 1 II-LiCl
(7oro oorc
e f
2 M-LiCl core 2 M-LXX CON>
#
2 x-IX1
cow
-~. 1 WLiCI split proteins 1 31.LiCl split proteins Purified thiostreptow binding protein (Bhl-LI
1)
i THS, [%]thiostrepton. Reconstitution of LiCl core pLtrt,icles with split prot~eins zrrrti :wsnys of the hio(lrnp ot’ /““Sjthiostrepton were carried out its described in Materials H~CI Methods. Each wsay contoinrd 50 prn(A of core particles or ribosomal subunits which were fini*lly incnbitted with 60 ~nnc~l [ “~S]thi~st,l.(~l)toll (430 cts/min per pmol). Reconstitution involved the ttdditiou of 100 1xnol equivalents of 1 a~.LiC’l split proteins or 100 pmol of purified protein BM-Lll (thiostrepton-binding protein) to 50 pmol of cores.
lines c to f; see also Highland et al.. 19756). Therefore: \I;(: decided t’o purify from 1 nl-LiCl split proteins the protein(s) active in restoring to 2 M-LiCl core particles the ability to bind the drug. (b) Purification
qf a protein
i,nvolved i?~ the bkding
of’ thiostreptot,
to ribosomrx
This was achieved by a two-step procedure involving ion-exchange chromatograph) on carboxymethylcellulose as the first step. The acbivt: component was retained on this column and was eluted by sodium acetate in the concentration range 35 t,o 43 mM (i.e. in the conductivity range 2.1 to 2.3 mmho; see Fig. 1). Active fractions were examined by electrophoresis on SDS/polyacrylamide gels and usually contained three or four proteins with molecular weights of approximately 28,000, 23,000, 17,000 (a minor component when present) and 15,500 (the major component). The second purification step involved chromatography on Sephadex GIOO in the presence of 6 M-UP%. Column fractions were again assayed for their ability to restore thiostreptonbinding activity to 2 M-LiCl cores (data not given). Fract.ions active in this assay contained essentially one protein as judged by electrophoresis on SDS/polyacrylamidc~ gels (Fig. 2(a)), urea/polyacrylamide gels at pH 4.5 (Fig. 2(b)) and polyacrylamide gels run in two dimensions (Fig. 6(d)). Th e molecular weight of this protein was determined to be about 15,500 on SDS/polyacrylamide gels and we estimated tha,t it was at leasb 9S”/h pure from the relative intensities of staining of major a.nd minor bands in these various gels. This single protein restored the ability to hind thiostrepton quantitatively when added to 2 M-Lick core part’icles (Table 1. line g). The minor contaminating component present in this preparation (see Fig. 2(b)) could not be removed and its nature is unknown.
THIOSTREPTON-RESISTAST
RIROSOMES
Fmction
1
241
no.
I“;
28000 23000 15500
f
FIG. 1. Partial purification from B. megaterium ribosomes of a protein involved in thiostrepton binding. 1 M-LicI split proteins from B. megaterkrz wild-type ribosomes were subjected to chromatography on carboxymethylcellulose at pH 5.6 in the presence of urea. Materials retained on the column were eluted with buffer containing a gradient of concentration of sodium acetate. Fract,ions ,)f the eluate were assayed for their ability to restore to 2 M-Lick core particles the ability to bind [%]thiostrepton (see Materials and Methods) and active fractions were analysed by eleotrophoresis on RDS/polyacrylamide gels. -O-O-, Thiostrepton binding; --a-e-, conductivity.
242 Mr markers
24 700
17 000 14 500 13 700
0~1 % SDS/ acrylamide
13% gel
El M-urea/ acrylamide
(a)
(c) Ident%@at%on
IO% gel
(pH4*5)
(b)
qf th,e
thiostrelltolz-Dinding
prolein
Having purified from ribosomes of B. meguterium a protcin \~~hich is principalI> involved in either creating or completing a binding sit,e for t~hiostrepton. n-c sought to identify it by immunological comparison with tjhe riboxomal proteins of E. coli. The thiostrepton-binding prot,ein of B. wzegaterium \las asscxsed by double immunodiffusion with each of the monospecific antisera raised against all 34 proteins from the 50 S subunit’ of E. coli ribosomes. X precipitin hntl WIS formed exclusively with an antiserum to E. coZi ribosomal protein LI 1. none of t hc ot’liw ;lrrtiswa g;rvcl ;w!precipitation (Fig. 3(a) : nega,tive rrsults not shown). Converscl~~. an antiswum raiwcl ‘against the purified R. megateriur~~ protein precipit,ated protcirl I, 1 1 alone and none of the other pure ribosomal proteins fkom the 50 S subunit of E. coli (Fig. 3(b)). These data clearly identif,y the thiostrepton-binding protein of B. megateriuat ribosomes as being homologous to pl,otein Lll of the E. coli ribosome. This pro&in was therefore designated BM-LI 1. anti E. coli Ll I formed a strong prucipitin band In a further series of experiments. with E. coli ribosomal protein Ll 1 but prccipitatrd. altwit, to a Icssrr dcgrw. ribosomal protein B&I-L1 1 of B. ~rrwgatwiuw (Fig. 3(c)). Spur formation Iwt~\wn tlw precipitin
243
I~‘IG. 3. L),,uble irnmunotliffusion. (a) Roaction of protein BM-Lll with antisera against rihosomul pr~kGns from thP 50 8 subunit of E. coli. The ccwtrc will c()ntamcd I pg HM-IAll. The peripheral w-PIIS rontaint : (I) anti-E. coli L7. y-globulin cxres1)ontling to 90 pl wrum-equivalents; (2) ;mtiE. co/i I& y-globulin (300 pl sc~lum-ccluivult~nts); (3) anti-E. coli LS. y-globulin (300 pl srrumequivdcnts) ; (4) anti-E:. coli LIO, 60 pl swum; (,5) snti-E. coli. Lll, 0.2 A,,0 specific antibody (purifktl ovw an h’. coli. LI 1 -:Ainit,y column); (6) arlt’i-E. coli. L12, y-globulin (90 pl serum-equivw lcnts). The ncgat,ivr results for the antisva against the- wmaining 28 ribosomal prokins from t,hc 50 S subunlt of E. coli RW not shown. (b) Reaction of anti-BWLIl with single ribosomal protrins from the 50 X subunit of E. coli. Th(> contw wc~ll contained anti-KM 1, f 1. y-globulin (1750 pl sc~~~~m-c~cluiv;~lent~s). The peripheral ~~11s contnnml (1) 2 pg E. coli. LlO; (2) Z pg E. coZi Lll; (3) 2 p,!q I<. coli LI”: (4) 3 pg E. coli L13; (5) 2 pg E. co&i L14; (6) 3 pg E. coli 1~15; (7) 2 pg E. coli 1‘16; (8) 2 pp E. coli L18. Except for L8, L17 anti LX1 the remaining ribosomal Irotrins from t,hP 50 S subunit of E. COG were tested in t,hc same manner‘ (data not shown).
bands indicat8ed that the proteins are homologous (i.e. scrologica~lly related) but not, identical (Fig. 3(c)). The converse experiment with antiserum raised against, II. WW~Iteriurrl BM-Lll gave a comparable result (Fig. 3(d)). The relatedness of the homologous proteins of the two bacterial strains was further corroborated by modified immunoelectrophoresis. R particularly sensitive technique. Thus (Fig. 4) an antiserum raised against protein BM-Lll formed a single st,rong precipitin band with total rihosomal proteins from B. w~yateriun~ and also with a lone rihosomal protein of E. coli which migrated exactly in the po&ion expected for
244
E.
CUNDLIFFE
E!t’
dL.
43
04
Origin J1
BM-LII
E. coli
L II
FIG. 4. Modified immunoelectrophoresis. 50 pg TP30 from E=. coli (l), 50 pg TP50 from E. coli (2). 25 pg TP70 from B. megaterium (3) and 60 rg TP70 from E. coli (4) were applied t,o the gel. E&&-ophoresis was for 80 min at 360 V and 3.8 mA in buffer d (SW Materials and Met,hods). Strip (b) was soaked in antiserum elicited against, BM-Lll prior to st,aining. Strip (a) was stained directly without soaking in antiserum. TP30, TP50 and TP70 refer to total prot,oins obta,inrd from 30 S rib{)somal subunits, 50 R ribosomal subunits or 70 8 ribosomw. respect~iwly.
protein Lll. It was also clear that protein Lll from E. CA’ and its counterpart (BM-Lll) from B. megaterium migrated differently during electrophoresis which confirms our conclusion that the two proteins are st’ructurally related hut not identical. The converse experiment to the one shown in Figure 4. but employing an antiserum raised against E. coli protein Ll 1. was a,lso performed (data not given) with similar results. We wish to emphasise here t,hat homology b&wren proteins Ll I of’ E. coli and BM-Lll of B. megatarium has been clearly demonstrated using antisera raised against either protein. The antiserum to protein LII was monospecific: and reacted exclusively with pure E. coli ribosomal protein Ll 1 and not with any other ribosomal protein of that organism (SMffler & Wittmann. 1971a.h: Stiiffler. 1974). Comparable experiment)s could not be carried out with anti-RM-Lll since individual ribosomal proteins from B. m,egatrrl;um are not yt+ available. Consequently, t,hc specificity of the antiserum to protein BM-Ll 1 has not’ been as thoroughly demonstrated as that of antiserum bo protein Lll of E. coli. However. the completeagreement between the results obtained with both antisera. described ht:rc, and below! revealed that the antiserum raised against prot)ein BM-LI 1 was also monospecific. The satellite bands visible in Figures 4 and 8 deserve further comment. They wcrt~ formed specifically with antisera raised against protein Lll or BM-Lll and have been seen also with ribosomal proteins from B. subtilis (Wiener1 et ml.. 1979). Since our antisera reacted with a single ribosomal prot,ein (viz. Ll I or its counterpart BM-L11 ) the satellites must, have been serologically related to protein Lll . Apparently, they represented a modified species of protfein Lll (or BM-Ll I ) although whether such modifications were physiological or artefactual remains to hc determined. However. we note in this context that E. c&i protein Lll contains three methylated amino acid
THIOSTREPTON-RESISTANT
RIBOSOMES
245
residues (Dognin & Wittmann-Liebold, 1977) and is the most heavily methylat,ed protein in t’he ribosome (Alix & Hayes, 1974; Chang & Chang, 1975) as is also t#he case for protein BM-Lll in B. megaterium (Cannon & Cundliffe, 1979). It is possible that the satellites represent differently methylated variants of proteins Lll and BM-Lll since differences in the degree of methylation of a protein might reduce. but \rould not be expected to abolish, its antigenicity. In fact, the non-methylated protein Lll from mutant prml of E. coli (Colson & Smith, 1977) was precipitat’ed by an ant’iserum to E. coli protein Lll (G. Stiiffler, unpublished data). (cl) Properties
of’ ribosom,es of thiostrepton-resistant
mutants
of B. megaterium
A number of spontaneously arising mutant strains resistant to t’hiostrepton were obtained from B. megaterium and three of them, designated PDI, PD14 and MJI. are described here. These strains which grow less rapidly t’han wild-type in the absence of drug are tolerant of much higher concentrations of thiostrepton than is the wildtype but are not totally insensitive to thiostrepton. When ribosomes and high-speed supernatant (SIOO) were prepared both from mutants and from wild-type and recombined in reciprocal fashion in cell-free systems synthesising polyphenylalanine, it became clear that the mutants owed their resistance to thiostrepton to some altered property of their ribosomes and that) their post-ribosomal supernatant fractions (SlOO) were not nota,blp different from that of wild-type (data not given). The synthebic activities of rihosomes from mutants PDI and MJI and from wildt’ype were compared in cell-free systems supplemented in each case with wild-type supernatant (SlOO). It was evident that ribosomes from either mutant were only about. 50(;!; as active as wild-type ribosomes even in t’he a’bsence of thiostrepton (Fig. 5). Also. ribosomes from the mutants were significantly more resistant, to the a,ction of thiostrepton in vitro without being tota,lly insensitive to the drug, a pattern which reflects the growth characteristics of these mutants. When ribosomes from mutant NJ1 (Fig. 5(b)) or mutant PDl (d a t a no t given hut similar to that in Fig. 5(b)) UXYY supplemented with protein BM-Ll 1 from wild-type. their synt’hetic activit,ies wew increased to wild-type levels and their resist,ance to thiostrept,on decreased dramat8ically. Similarly, whereas ribosomes from all three mut’ants bound [ 35S]thiostrepton poorly in comparison with t,hose from the wild-type. they did so quantitat’ively when supplemented eit’her wit)h 1 M-l,icl split) proteins from wild-type or with purified prokin BM-Ll 1 (Table 2). (c) Elwtroph)oretic
&di~s
?f rihosomal
proteinn
of thiostrepton,-resistant
mutants
The ribosomal proteins of mutants MJl, PDI and PD14 were compared with t,hostk of \viId-type B. megaterium, by two-dimensional polyacrylamide gel electrophoresis. ‘t’h(~ pa,tt’rrns of spots revealed after staining proteins in the gels were essentially icknt’ical with one not’able difference: the spot corresponding to protein BM-Lll in wild-type (Fig. 6(a) a’nd (d)) was missing from the gels containing ribosomal prot’eins from mutants MJl or PDI (Fig. 6(b) and (c): data for mutant) PD14 not given). Also, the absence of this spot was not accompanied by the appearance of any “exka” spot’ which would have revealed the existence of a mutationally altered protein migrating differently during electrophoresis. The ribosoma,l prot,eins of the three mutants have also been compared with those from wild-type B. megaterium 1, cellulose acetate gel electrophoresis using three different buffer systems (see Materials
240
Wild-type
Plus plus &==-I-@0
Mutant
ribosomes
or minus THS
MJI
ribosomes
BM-LII, I
20 Time
(min)
Ribosomss (50 pmol) WCI‘P incubut,ocl at, 0 ‘C untl at 20 C’ with ~tntl without :I pg of’pr~~tein B&L 11 (see Mat&& anti &lMothods). Sometimes, thiostrepton (:300 pmol) wits then art&xl and incuhat,ir)n continued at 20°C’ for a further 5 min. Synthetic activity of ribosomc>s was then asrayecl at, 37 ‘(’ following addition uf high-sprrd sup~rnatant (5100) from I{. rr~grcteric~,rn wiltl-t~ypcs, t,ogethcr with cocktail oor&aining (l*C ]phonylalanine. polg(U) etc. (WC Mt~t~rials and Methods). Samples (10 ~1) were withdrawn from incubation mixtures, placed in 10~~~ (w/v) t&hloroacetio acid, heatetl at 90 Y’ for 30 min and pr~~ss~~l RX described in Materials ant1 Mlrthotis. Thiost,rept,on (THS) was tlirsolvod in tlimet,hyl rulphoxitk which with ILIW ;trlcIc(l to cvlnt?oi I II cubations at a final concentration of 1.25”i, (v/v). 1 pmol [ 14C]phmyla1anint~ gavr approximtrtc:ly , ribosomcs plus 1000 cts/min under t,hhetir conditions. (- a--(: -, 1Zibosomcrj ;tlorw ; -l,.protein B&I-L11 ; -@----a-, riboaomcs plus thug: --I--& , riboxomt,s l;ius lrotein B&l-1~11 plus drug.
TABLE
Binding Rourne (50
qf 35X lthiostrepton of ribonomrs pmol)
2
to rihosolnrs Atltlitions
of B. megaterium [ “5Nlthioxtrrl)tolr hountl (17m01)
Ribosomea (50 pmol in RS buffer) were incubated for 5 min at O“C then at 20 (’ Lvith t.itht.1 100 pmol of prot,ein BM-Lll from R. megateriune wild-t,ypcx or with 75 pmol equiv&nts of 1 nr-IX1 Rplit proteins from wild-type. Then [93]thiostrepton (60 pmol; 250 cts/min per pmol) was otcltietl and incubation continued at 20°C for a furthor 15 min. The extent of binding of drug to rihosomvs was then assayed by go1 filt8r&ion (see Materials and Methods). f wt, wild-typo.
THIOSTREPTON-RESISTANT
247
RIBOROMES
lb)
Frc:. 6. Analysis by 2.dimensional polyacrylamide gel electrophoresis of tot,al ribosomal prokin* from 11. megnterium wild-t,ype and mutants, and of purified protein BM-Lll. The gel system was based upon that of Kaltschmidt & Wittmann (1970) as described in Mat~crial~ and Methods. Elecfrophoresis in the 1st dimension (pH 8.5, 4% ( w ./ v ) acrylamide) was curried out at 4°C for 12 h at 300 V anode-to-cathode for basic proteins and for 4 h at 150 V (also at 4°C’) cathode-k-anode for acidic proteins. Electrophoresis in the 2nd dimension (pH 4.5, 18% (w/v) acrylamide) was carried out at 4°C for 20 h at 120 V anode-to-cathode. Proteins in the final slab gels were stained with Coomassie Brilliant Blue G250 (0.04?4 (w/v) in 3.5% (w/v) perchloric aciti) and were destained using a mixture of 15% ( v / v ) mrt,hanol and 7.5% (v/v) glacial acetic acid. So differences were detected between the acidic ribosomal proteins of the wild-type and any mutants and, for ease of presentation, data relat,iug to acidic proteins are not given in Pig. 6. (a) Basic ribosomal proteins from N. megntwium wild-type; (b) basic ribosomal proteins front mutant MJl ; (c) basic ribosomal proteins from mutant I’D1 ; (d) purified prot,oin BML11 (thiostr~:l,ton-~,intling protein) from wild-type.
and Methods). It was found that a single protein-band st’rain. was absent in each of the three mutants (data The dat*a presented in Table 2 and in Figures 5 and 6 from mutants MJl, PDl and PD14 either lacked protein counterparts of BM-1~11 which co-electrophoresed with from which they could not be distinguished on standard resolved by examining the ribosomal proteins of the w&h wild-type by immunological techniques.
which is present in t’he parent not shown). clearly indicate that ribosomes BM-Lll or possessed variant, some other ribosomal prot,ein(s) gels. These possibilities were mutants and comparing them
E.
248
(f) Immunologicnl
properties
CUNDLIFFE
ET
of rihosomal
A I,
pofei1r.v
of ihiostrrl,torl-rp.si.st(~llt
rnutatrt.s
Total ribosomal proteins from wild-t,vpr, H. r//e@eriurrr aud from muta,nts t’l)l. PD14 and MJl were examined by immunodiffusion with antiserum raised against ribosomal protein Lll of E. COG (Fig. 7(a)). Proteins from the wild-type produced w weak precipitin band whereas those from the mutants did not. These initial results supported two contentions: namely, tha.t the protein altered or absent in the mutants (as compared with wild-type) is related to E. COG protein Lll and t,hat ribosomes of
(a)
(b)
FIG. 7. Double irnmunodiffusion oxlxximent~a with (a) The centre wall cont,ainod 250 pl anti-E. coli Lll. TP70 from H. meycslerium wild-type; (2) 60 pg and (3) PDl; (4) and (5) blank; (6) 100 pg TP70 from IY. eoli: (b) The centre well contained 260 pl anti- /I. megrcteriurn TP70 from B. meycrterium wild-t,ype.
(1) 100 pg TP70;
(2) 150 pg TP70;
ribosomal lw&eins from mutant 1’l)l. Tho peripheral wells wnt,aincd: (1) 60 pg IN pg TP70 from tl. rnogtrteriz~m mutjant (7) and (8) bla,nk. HM-LI 1. Thv priphwal w~~lls rontarnt~l
(3) 200 pg Tl’iO.
the mutants do not contain any protein able to be precipitated by t,he ant,iserum raised against protein Lll of E. coli. This conclusion was further substantiated using the antiserum against protein BM-Lll which reacted more strongly with the homalogous antigen in ribosomes of B. megateriumn wild-type and could therefore give morci rigorous evidence as to whether or not the ribosomes of the mutant,s contained protein BM-Lll. Again, ribosomal proteins from the wild-type but not Ohose from the mutantti formed precipit’in bands with this antiserum (Fig. 7(b) and (c)). This result. strongly suggests that ribosomes from the mutants do not contain ribosomal protein BM-I,11 . However, since negative immunochemical results cannot always bc regarded as conclusive. we attempted to confirm these result’s using a, more sensitive technique. namely modified immunoelectrophoresis (Fig. 8). This method is not simply based on immunoprecipitation but also on binding of specific antibody to the antigen which is immobilized on a cellulose acetate gel (Zubke ~1 al., 1977 ; G. Stiiffler t R. Ehrlich. unpublished results). This technique has proved to be about an order of magnitudtx more sensitive than Ouchterlony double immunodiffusion in det,ecting strong antigens and has even allowed the detection of certain weaker antigens (e.g. fragments of ribosomal proteins) which do not form precipitin bands with antiserum during double immunodiffusion (R,. Ehrlictr & G. Stiifflcr, unpublishad data). Again (see also
THI08TREPTOS.RESISTrZXT
249
RTBOSOMES
e
oe
Origin J
f BM-LII 25 pg (1 anti 26 pg (4), 37.5 Elcrtmphorcsis Prior to staining
7) ad 12.5 pg (2 anti 8) of TP70 pg (5) and 50 pg (6) of TP70 from was for 100 min at 250 V and 3.7 the strip was soaktd in antiswum
of
11. megtctarium wild-type; and 12.5 pg (3) II. megrrterium 1’111 were applied to the gel mA4 in buffer e, (SW Materials and Methods) clicitetl against B&l-Ll 1.
Fig. 4(b)) a strong precipitin band plus a weaker sa,tellit’e was obtained with B. megateriurn wild-type ribosomal proteins in response to antiserum specific for protein BM-Lll. In contrast,, no precipitin ba,nds were obtained wit’h ribosomal proteins from any of t#he three mut’ants even when a’ broad range of antigen concentrations was assessed. We therefore concluded that we had eliminated all but t,he remotest possibility that any cross-reacting material related to protein BM-Lll was present on t*he ril)osomes of mutants MJl, PDl and PD14.
4. Discussion Ribosomes of Bacillus megaterium contain a protein (BM-Lll) which is immunologically related to protein Lll of E. coZi ribosomes. These two proteins have similar molecular weights, namely 15,500 as estimated for protein BM-Lll (Fig. 2(a)) and 15,300 as estimated for protein Ll 1 by a similar technique (Zimmermann t Stoffler. 1976) and are further related by their ability to create or complete a ribosomal although neither protein binds thiost#repton off the binding site for thiostrepton, ribosome. Proteins Lll of E. coli and BM-Lll of B. megaterium are also related in that they are the most heavily meth,vlat,ed prot’eins in their respective ribosomcs (see above). Although no biological role yet postulated for protein Lll has been related to the fact that it is methylated, the protein itself has been implicated in various aspects of ribosome assembly and function. Thus, protein Lll binds to 23 S RNA in, vitro under condit’ions where t,he protein is not denatured (Lit~tleahild rot al.? 1977) and is
260
E.
CUNI~LIFFF,
ET
A L.
involved in integrating protein Ll6 into t,hc peptidyl t,ransferaw centjrc oft tw ribosonw (Diet)rich it ccl., 1974; Moore et al.. 1975). ItI has also been sugg&ed t,hat protrin I,1 1 is crucial for the expression of peptidpltralisf~rase ac!t,ivit?, (Nierhauw & ?dOtlt~fb,~fJ. 19%‘) but, this conclusion is hardly compat’ibk, \vit,tt our data. since l~ihMCJlllC~S ot mutants PDl, PD14 and MJl which lack protein BM-LlI are act,ivr in prot,t+n synthesis i7~ vitro (see Fig. 5). Other investigators have also shown that, protttin I,11 is not obligatory for peptide bond formation (Howard & Gordon, 1974: Ballesta $ Vazquez, 1974: Moore et al.. 1975 ; Armstrong & Tate. 1978). Prot,ein Lll is lab&d by a photoactivated analogue of GDP in the presence of elongation fa,ctor EF-G (Maasen & Miiller, 1974) and has even been postulated to be t)he ribosomal GTPaw enzyme (Schrier & Miiller, 1975). Cross-links have also been formed between pr&oin Lll and proteins L7, L12 and L10 (Expert-Bezanqm rf al.. 1976) and also proteins L2, L4 and L14 (Kenny & Traut, 1979). These proteins arc in the neighbourhood of the ribosomal binding domains of the protein synthesis fact,ors IF-2, EP-Tn. EF-G. RF-1 and RF-2 and are close to the site where GTP hydrolysis occurs (for reviews see Miiller, 1974; Miller & Weissbach: 1977: Brat,, 1977). Mutants of E. coZ?Iwith variants of protein Ll I have been isolated and have in some cases (reZC) been shown t’o exhibit relaxed control of RSA xynt’hwis (P>lrk(lt of al., 1976). Sinor thiostrepton also inhibits thrl ri~)osonlc,-tlcp(,I~~~~,rlt formatiotl of’ guanosinc t,c+a- and penta-phosphates in extra& of stjringc~ntj (~11s (Hawltiw A r/l.. 1972 ; Pederscn rf al.. 1973) in addition t)o exerting t htx other c+Yccts outlitwtl t~at~lit~r. thcrc is a substantial correlation between post,ulated functions of prot,cLiu Ll I antt processes inhibited I)y thiostrcpt,on. Howevrr. in viw of our obs~rv;~tion t tlat mutants MJl. PII1 and PD14 possess func~tional ribosomcs \vhich 1ac.k ~~rotckr BM-Lll, no indispensable function can be attributcld to this protein (or t)y inti~t~c~tlc~c~ to protein Lll of E. coli). This swvcs to tmphasiw thr itnportaticc of cw-c)pcwti\-ts interactions bet,\veen ribosomal components in thrl cxprcssioti of their functions. Our dr>monstration t,hat ribosomes frotn muta&s M,Jl ant1 I’DI (resistant to thiostreptjon) are restored to lvild-type levels of act,ivity and drug scansit ivit y \\.trrw supplemented wit,11 prot’cin BM-Ll 1 might have bwn caxplioa 1)1(xin terms of r~xc+hatryc~ of proteins between the ribosomc-bound and free forms. particularly sitrw proteitt Ll 1 of E. cnli has been shown to undergo such cxchangc ire ,vitw (Rohrrtson c/ (41.I 1975: Subramanian & van Duin. 1977). WC therefort: felt ii- (~ncutnlwnt upotl 11s to establish b!- t,he most, rigorous means possible that rilwsotrrcs ftwtl out’ tnutatlt s atv deficient in protein HIV-TA 1. It is quite difficult to establish unambiguously \\~hethcr ot’ not’ a protcliu is missing from ribosomes. The difficulties in reaching such a conclusion in the (:atit’ of wrcral E. coli mutants which possibly lack protein 820 ww t,xtcnsi\-oly discuswd 1)~. Witjttnann nf al. (1975). For t’his reason we exatnined t)hc\ ri hosotnal prot’c+ns of out’ mutants I)y WVtbrill c~lcctrophorot,ic and irnmutloohc~mici~l tI&niques. Ttith twults obtainctl wtw in complctc accord and indicated t’hat prot,cin BM-I,1 1 is missing from the ribosomcs of our thiostrept,on-resistant mut,atits. t n particular. wheti ttlodificti immutioclt~ct~ropt~oresis \vas employed for a rigorous c~xwminatioti of the t~ihS~~I~li~l proteins of mutants PDl. PD14 and MJl~ no cross-reacting tnatt~tial rc~latetl to eit,hrr H. megatprium protein B&I-L1 1 or E. coli protein LI 1 \vas detected. W’V COIIsider this data as being most convincing. Accordingly, wc conoludo that) no countwp;l rl of prot’ein BM- Ll 1 whether drastically altered or undt~rmet~1yI;lt~ecl or evcm an ‘.a trtltc~t~” fragment,, \\‘as present, on the rihosomcs of the t trrtw mut,ants studied.
‘I’HIORTHF:I’TON-IZE:SIHTANT
RIHOSOhIES
2.5 I
We then sought to establish whether cross-reacting material related to protein BN-IAl was present elsewhere in our mutants. Conceivably such material might not ha,vcx INXI~ assembled into ribosomes of the mutants or might have been lost from the ribosomes during their preparation. The latter case is perhaps unlikely since residual traces of protein BM-Lll should have been detected on t,he ribosomes. Preliminar) titudies revealed such cross-reacting material in acetic acid extracts from total cells a,nd in the SlOO fraction. Analysis by double immunodiffusion revealed only pnrt’ial serological identity between protein BM-Lll and the cross-reacting material as judged from the formation of a spur. We are unsure of the nature of this cross-react,ing material and further studies will be needed to characterise it. However, our abilit’y to detect such cross-reacting material among the total cellular proteins of the mutants proves that t,he modification(s) introduced by mutation did not prevent prc:cipitation of some form of protein BM-Lll bg the antiserum. This reinforces our conclusion that’ had a,ng such material been present on the ribosomes of the mut’ants we should have detected it. Results similar t,o some of those report,ed here have also been obtained indepcndent]?; and concurrently by others. Thus, certain mutants of B. suOtiZis(Goldthwaite & Smith, 197%; Pestka et al., 1976: Wienen et nZ., 1979), arising spontaneously and resist’ant to thiostrepton, possessed properties somewhat similar to those of mutants I’DI. PI)11 and MJl. Furthermore, wild-type B. mbtilis cont’ains a ribosomal protein ES-I,11 \j.hich is relat,ed to E. coli protein Lll and to BIN-11 bot,h immunologicall! ittld in its allility to promote the binding of thiostrepton to ribosomal core particles (N?clnen et al., 1979). Six t’hiostrepton-resistant mutants lack protein BS-Lll ; the protcitl ~~1s found t,o recur in six spontaneously-derived rev&ants (Wienen et al.. 1979). ‘I’llis \vork TVASsupport)ed by grants from t,he Deutsclle E’orsclturl#s~es~~llscllaf; (to U.S.) at~d t Ilrs Medica. Research Council (t,o E.C.). We are also indebtetl t,o MRC for Researclt Strlctcwtslrips (for P.D. and M.S.) and to Professor H. G. Wittmanrr and Dr M. J. Dognit for pro\-iding puw E. coli rihosoma,l proteins. The patient criticism of E. R. Dabh ant1 B. \\‘ic~netl is gratefully acknowledged. I)r C’. (‘olson kindly provided rnlltantj ,urml of E. co/i.
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