identification of neighbouring proteins in escherichia coli 30 s ribosome subunits

J. Mol, BioL (1977) 112, 603-629

Identification of Neighbouring Proteins in Escherichia call 30 S Ribosome Subunits

A. EXPERT-B~zANqOm, D. BARRITAULT, M. MILET, M.-F. GU~RIN ~ D D. H. HAYES

Laboratoire de Chimie Cdlulaire, Institut de Biologie Physico-Chimique Fondation Edmond de Rothschild, 13 Rue Pierre et Marie Curie

75005 Paris, France

(Received 6 February 1976)

A large number of cross-linked protein complexes are formed in widely varying yields by treatment of Escherichia cell 30 S ribosome subunits with bisimidoesters. Methods for the fractionation of the protein complement of bisimidoester-treated 30 S subunits, the isolation of cross~hnked complexes, the cleavage of bisamidine cross-links and the identification of released proteins are described. Thirteen complexes produced by treatment of 30 S ribosome subunits with dimethyl suberimidate are identified. When shorter reagents are used, ten of these com. plexes are formed with dimethyl adipimidate and two are formed with dimethyl succinimidate.

1. I n t r o d u c t i o n

Information concerning the spatial relationships between individual proteins in the intact ribosome can be obtained by isolating and characterizing the products formed by the action of bffunetional protein-specific reagents on ribosomes or their subunits. Several recent publications describe the use of a variety of cleavable and non- cleavable cross-linking agents for this purpose (bisimidoesters: Bickle et al., 1972; Clegg & Hayes, 1974; Lutter eta/., 1974a; Barritault eta/., 1975a,b; Expert-Bezangon et al., 1974; Peretz et at., 1976; bismaleimides: Chang & Flaks, 1972; Lutter et al., 1972; bisazides: Lutter et al., 1974b,1975; tetranitromethane: Shill & Craven, 1973; mercaptobutyrimidate: Traut et al., 1973; Sun et al., 1974; Sommer & Traut, 1974a,b, 1975,1976). In this paper we give details of procedures for the isolation and characterization of cross-linked complexes formed by the action of three bisimidoesters of increasing chain length on 30 S ribosome subunits. The nature of the structural information obtained in this way and the limitations of the cross-llnl~ing technique are discussed. Preliminary accounts of this work and of related experiments with Escherichia coli 50 S ribosome subunits have already been published (Barritault et al., 1975a; Expert-Bezan~on eta[., 1974).

2. Materials and Methods

E. cell MRE600 was obtained from Whatman (England) as a frozen eel] paste. Suceino., adipo-, subero- and sebacodinitriles obtained from Eastman Kodak (U. S. A.) wore used for the preparation of bisimidoesters (Davies & Stark, 1970) without purification. All other chemicals were reagent grade products supplied by Prolabo, France; Merck, Germany; Eastman Kodak, U.S.A.; or Fluka, Switzerland.

603

604 A. E X P E R T - B E Z A N ~ O N E T A L .

RPS-X-0mat ~]m for autoradiography was a product of Kodak, France. Carrier-free [~6S]mflphurie acid was purchased from the Commlasariat ~ l'Energie Atomique, l~rance.

Ca) Buffer8 and reagen$ solutior~s (1) 0-1 :~-trietbanolamlne .HC1 (pH 7-2), 0-01 M-MgC12, 0.05 M-KC1, 0.006 ~-fl-mercapto-

ethanol. (2) 0.01 M-triethanolamine. HC1 (pH 7-2), 0.0I M-MgCI2, 0.05 M-KC1, 0.006 ~-fl-mercapto-

ethanol. (3) 2 ~.triethanolamlne, 0.05 M-KC1, 0-01 M-MgCI 2. (4) 0.01 M-triethanolamine.HC1 (pH 7.2), 0.5% (w/v) sodium dodecyl sulphate, 0.01 M-

~-mercaptoethanol. (5) 0.1 ~-triethamolamine-HCl (pH 7.2), 0.01 ~-MgC12. (6) 0.01 ~-triethanolamine-HC1 (pH 7-2), 8 ~-urea.

(b) Labelling of bacteria

[35S]sulphuric acid (1 mCi/ml) was added to early logarithmic phase cultures of E. coli MRE600 growing in a meditun containing a low concentration of sulphate (Clegg & Hayes, 1974), and growth was allowed to continue until a maximum of 50~o of the radioactivityt had been incorporated. Bacteria were harvested by centrifugation.

(c) Preparation of ribosomes and ribosome 8ubunits All operations were carried out at 0 to 4°C. Bacteria were disrupted by grinding with

alumina (ssS-labelled cells) or by the use of a French pressure cell (unlabelled cells), S 30 extracts were prepared in 0-01 M-Tris.HC1 (pH 7-4), 0-01 M-MgCla, 0-05 M-NH,C1, 0-006 ~- fl-mercaptoethanol and their ribosome contents were isolated by centrifugation (3 h at 150,000g) as previously described (Expert-Bezangon e$ al., 1974). Ribosomes were dis- soeiated by suspension in 0.01 ~-Tris.HC1 (pH 7.2), 0.01 M-MgC12, 0.4 M-NaC1, 0.006 M- fl-mercaptoethanol and subunits were separated by sedimentation of the suspensions in sucrose gradients prepared in the same buffer (85S-labelled ribosomes: 25 ml, 5% to 20% gradients, rotor SW25.1, 16 h at 20,000 revs/min; unlabelled ribosomes: 1600 ml, 7% to 26% gradients, zonal rotor B15, 17 h at 24,000 revs/min) and concentrated from pooled gradient fractions by precipitation with 10% (w]v) polyethylene glycol. Full detaSls of these procedures are given in an earlier report in which we show that subunits prepared by dissociation of ribosomes in the presence of 0.01 ~-Mg 2 + and 0-4 ~-NaC1 are several-fold more active, as measured by their capacity to catalyse lysozyme synthesis in vitro with bacteriophage T4 messenger RNA as template, than subunits prepared by dissociation of ribosomes in the presence of 0.1 mM-Mg 2+ (Expert-Bezangon et a/., 1974). The specific activities of preparations of 36S-labelled 30 S subunits varied from 2 X 106 to 5 × 106 ors/rain per O.D. unit at 260 nm.

(d) Prepara$ion and 8Scrags of bi~imidoes~rs Dihydrochlorides of dimothyl succinimidate, dlmethyl adipidimldate, dimethyl suberi-

midate, and dimethyl sobacimidate prepared by the method of Davies & Stark (1970) showed no significant loss of activity during prolonged storage (at least 3 months) at 4°C in vacuo over silica gel. The first 3 of these compounds were characterized by elemental analysis (dimethyl succin~midate, found: C, 34.0; H, 6'4; N, 15-4; C1, 29.7; calc. for CeH14N2OaC12: C, 33.19; H, 6"50; N, 12.90; C1, 32"66; d~methyl adipimidate, found: C, 39.3; H, 7.7; N, 11.5; C1, 29.1; calc. for C6HlsN~O2CI2: C, 39.19; H, 7.40; N, 11.42; C1, 28.93; dimethyl suberimidate, found: C, 44.0; H, 8-4; lq, 10.3; C1, 26-0; sale. for Cx0H22- N20~C12: C, 43.96; H, 8.12; N, 10.25; C1, 25.95) and melting point determinations. Upon heating, the bisimidoester hydrochlorides decompose with formation of the corresponding diamides which in the case of succinamide is decomposed further to sucelnlmlde (Roger & Nielsen, 1961); the melting points observed for the bisimidoester hydrochlorides are therefore those or close to those, of these decomposition products. Dimethyl succin~m~date,

t Some samples of commercial [ssS]sulphuric acid have been found to contain variable amounts of non-incorporable radioactivity.

N E I G H B O U R I N G R I B O S O M A L P R O T E I N S 605

tm =- 90 to 96°O (suooinamide, gm ---- 260°0; suocinimlde, t m --~ 126°Ct). The melt ing po in t of d lmethy! sucoinimidato has been previously repor ted as "abou t 98°C ' ' (Havsteon, 1973).

Dimethyladip imidate , t~ ~ 220 to 222°C (adipamide, t~ ---- 220°Ct). DMS~:, t~ ----- 215 to 217°C (suberamide, t m = 216 to 217°C~). The tm value of DI~IS was previously repor ted as 216 to 217°C (Davies & Stark, 1970).

(e) Reaction of ribosom6 subunits with bisimido~t~ra

Immedia t e ly before use bisimidoester dihydrochlorides were dissolved a t a tenon of 0.15 M in a miYture of buffer (5) and 2 ~ - K O H (85:15, v/v). The p H of a suspension of 30 S r ibosome subunits (Aa0o am ---- 10) in buffer (2) was ad jus ted to 8.2 b y addi t ion of 0.06 vol. reagent solution (3); 0.10 vol. of the neutral ized solution of bisimidoestor was then added (final bisimldoester oonen approx. 0.015 ~ , about 100 molecules of reagent per free prote in amino group) and the mixture was incubated a t 30°C for 90 rain. 30 S subuni ts were then prec ip i ta ted b y addi t ion of a 50% (w/v) solution of polyethylene glycol (Carbo- wax 6000) in buffer (2) to a final concn of 10% (w/v) (Expert-Bezangon et aL, 1974), collected b y oontrifugation, and resuspended in buffer (2). Their prote in and R N A components wore dissociated b y addi t ion of 0.1 vol. 1 M-MgCI2 and 2 vol. of glacial acetic acid to the suspension (Waller & Harris , 1961 ; H a r d y et al., 1969). R N A was removed b y oentri- fugation and proteins wore prec ip i ta ted from the supornatant b y addi t ion of 5 vol. acetone (Barr i taul t et aL, 1976a) and recovered b y contrifugation. The prote in pellet was held briefly in vacuo (water pump) to remove acetone, and dissolved in 0.1 ml of buffer (6). Prote ins wore roprocipi tated by addi t ion of 5 vol. acetone to remove remaining traces of acetic acid, recovered by contrifugation, dr ied in vacuo, and redissolved in 8 M-urea, 0.1 ~- /Lmorcaptoothanol a t a conch of 30 to 50 mg/ml.

(f) Electrophoretic fractionation of lrrot~ins isolated from bisimidoezter.treated 30 S ribosome subunits

Samples of prote in prepara t ions obtained as described above (200 to 300 #g, 108 ors/rain of 35S) wore f ract ionated by 2-dimousional electrophoresis in a horizontal cell as described elsewhere (Barr i taul t et al., 1976b) using polyacrylamido gels and buffer systems with the compositions described b y Ka l t sehmid t & W i t t m a ~ n (1970) modified b y using 15% (w/v) aorylamide, and 0.25% (v/v) bisaerylamido in the second-dimension slab to improve the resolution of higher molecular weight material . Af ter olootrophorosis gel slabs were sta£ned with Coomassio bri l l iant blue R250, dr ied in vacuo a t 50°0 (Barr i taul t et ed., 1976b), and radioact ive spots were detected b y contact autoradiography.

(g) Isolation and characterization of cross-linked protein complexes

Isola t ion: radioact ive spots corresponding to cross-linked complexes wore cut out of dr ied gel slabs and their aaS content quant i ta t ive ly eluted b y incubat ion a t 40°C in 0.5 to 1 ml of buffer (4), for 48 or 72 h (complexes wi th es t imated molecular weights ~30,000 and > 30,000, respectively). A to ta l of 300 #g of unlabelled to ta l 30 S proteins were added to each eluate and labelled and unlabelled proteins were allowed to precipi ta te overnight af ter addi t ion of 5 vol. acetone (Barr i taul t et al., 1976a). They were then collected b y centrifugation, dr ied in vacuo (water pump) and redissolved in 200 ~1 of 10~/o (w]v) sodium dodecyl sulphate, 0.2 ~-fl-mercaptoethanol.

(h) Molecular weight mex~sursmsnt~

Samples of the solution of olution products containing a t least 2000 ors/rain of 35S wore analysed b y electrophoresls in ver t ical polyacrylamide gel slabs in the presence of sodium dodecyl sulphate according to the 1-dimensional procedure of Laemmli (1970) or, in some cases, b y the use of the 2-dimensional technique of Mets & Bogorad (1974) as modified b y Subramanian (1974) for appl icat ion to E. co / / r ibosomal proteins. I n bo th methods a

t Melting points are quoted from Rodd (1952). Abbreviation used: DMS, dimeghyl suberimidate.

606 A. EXPERT-BEZANQON ET AL.

mixture of haemoglobin monomer and oligomers, prepared by treatment of haemoglobin with DMS as described for ribosome subunits, was used as a source of standards for calculation of molecular weights.

(i) Determination of protein compositions After removal of samples for molecular weight measurements the remaining solutions

of elution products were mixed with 4 vol. of 6.5 K-NH4OH containing 1.0 K-acetic acid (final sodium dodecyl sulphate concn 2%, w/v) and the mixtures were incubated at 30°C for 15 h to cause ammonolysis of imidoester cross-links. Proteins were then recovered from the reaction products by precipitation with 5 vol. acetone. Centrifugation of the resulting mixture caused separation of 2 phases, and precipitated proteins were found in a pellet and in a membrane at the interface of the 2 phases. The latter was broken with a glass rod and the membrane fragments were collected by centrifugation. The resulting protein pellet, which sticks slightly to the surface of glass centrifuge tubes, was freed of ammonium hydroxide and acetic acid and dried by washing with 3 1-ml vols acetone. it was then redissolved in 100 pl of 8 K-urea, 10- 2 K-triethanolamine (pH 7.8) and proteins were reprecipitated by adding 500 pl acetone, and dried at a water-pump vacuum. The final pellet was either stored dry at --20°C or dissolved for analysis in 10 pl of 8 m-urea, containing 0.1 ~/o (w/v) bromoplienol blue and 0.1 K-~-mercaptoethanol. After incubation of the mixture at 40°C for 10 rain, 2-dimensional cleetrophoretic analysis of ammonolysis products was carried out (Barritault et al., 1976a) using buffers and polyacrylamide gels with the compositions described by Kaltschmidt & Wittmarm (1970). After electrophoresis, gels were stained, dried and autoradiographed. Stained spots corresponding to carrier 30 S ribosomal proteins and spots visible in the autoradiographs were cut out of the dried gel slabs and dissolved by incubation for 15 h at 50°C in 0.2 ml of 50% (v/v) H202. The 35S content of the resulting solutions was measured after additon of 1-5 ml of Triton Xl00]toluenefPPO/POPOP scintillation fluid.

3. Results

(a) Characteristics of bisimidoesters and kinetics of cross-linking and ammonolysis reactions

The structures and dimensions of the four bisimidoesters used in the work described here, and the reactions of formation and ammonolysis of bisimidate cross-links and of bisimidoester hydrolysis, are shown in Figure 1. The kinetics of cross-linking by DMS under the reaction conditions defined in Materials and Methods were determined in a model system in which [3H]Lys-tRNA T M was at tached covalently to 70 S ribosomes by treating [3H]Lys-tRNA-poly(A)-70 S complexes with this reagent. The data obtained (Fig. 2) show tha t the reaction measured in this system, i.e. cross- ]int~iag of lysine residues carried by Lys- tRNA to ribosomal proteins, is complete in two hours. We consider this result to be an indication of the probable rate of formation of interprotein cross-ilnl~s during t rea tment of ribosome subunits with DMS. To determine the l~inetics of ammonolyt ic cleavage of imidoester cross-links, a sample of a cross-linked complex containing 30 S proteins $5 and $8 (complex G, see below) was subjected to ammonolysis under the conditions defined in Materials and Methods, samples of the reaction products were removed after increasing times of incubation and their content of unchanged complex and of released proteins $5 and $8 was determined (similar results were obtained in experiments carried out with complex F1, i.e. $7-$9, see below). Inspection of Figure 3 leads to the following conclusions.

(1) Ammonolysis is accompanied by an initial rapid decrease in the amount of the input radioactivity which is recoverable in the form of identifiable cleavage products (proteins $5 and $8) and unreacted complex G (Fig. 3). Because of this decrease only

N E I G H B O U R I N G R I B O S O M A L P R O T E I N S

Bisimido esters

f l y s i n e Protein 1 terminal Protein Protein

I I I H3C---O--C---(CH2),--C---O--CH3 + NH2 -~ 2CHvOH + HN--C--- (CH2) , - -C-- -NH

II II II II N + N + N + N + Ha Ha H~ Ha

= 2 Succinimidate d = 3.78 A. = 4 Adipimidate d = 6-3 A = 6 Suberimidt~te d = 8-8 J~

~ = 8 Sebacimidate d = 11-3 J~

6 0 7

Ammonolysis

Protein Protein Protein

I I I HN--C---(CH2)n--C---NH + 2NH3 -~ 2NH2 + H2N--C--(CH2) , - -C-- -NH2

H II II II N N N N H H H H

Hydrolysis

H3C---O--C-- (CH2)n--C--O--CH~ + 2H20 -* H 3 C - - O - - C - - ( C H 2 ) , - - C - - O - - C H ~ + 2NH +

II IJ II II N + N + 0 0 Ha H2

FIO. 1. Structures and reactions of bisimidoesters.

u~

U > ,

>

Q~

I0

9

8

7

6

5

5

I 0 0°5

I I I I I I ] '5 2 2 . 5 3

Time (h)

FIG. 2. Kinetics of covalent binding of [SH]Lys-tRNA to 70 S ribosomes by reaction with DMS.

Lys- tRNA-70 S complexes were formed by incubating [3H]Lys-tRNA ([SH]lysine-charged total E. coZi t R N A containing 55 pmol [aH]Lys-tRNA, spee. act. 1.5 × 10 e cts/min per pmol) ; E. coZi 70 S ribosomes, 550 pmol; and poly(A), 12 pg, for 10 rain a t 30°C in 500 pl of 0.1 wr-tri-

4O

608 A . E X P E R T - B E Z A N ~ O I ~ I E ~ ' ~IL.

about 40% of the input radioactivity is recovered in identifiable products after completion of ammonolysis. This loss of material can be accounted for in par t by the fact tha t the recovery yields shown in Figure 3 are based on measurements of the total radioactivity eluted from zones containing unreacted complex G and released proteins $5 and $8, excised from polyacrylamidc gels in which samples of reaction mixtures were analysed by one-dimensional analysis. These yields therefore take into account loss of material associated with elcctrophoresis (retention in the starting zone, etc.~ and with the elution process. Since only 65 to 70~/o of the input radioactivity of samples taken at zero t ime or after 15 minutes incubation is recovered after electrophoretic analysis, these factors account for at least 50~/o of the total loss of radioactivity observed after ammonolysis of cross-linked complexes. Peptide bond cleavage, and the consequent generation of small protein fragments, may also be a contribu- tory factor. This is suggested by the observation of small amounts of radioactivity moving in a diffuse zone with a higher elcctrophoretic mobility than proteins $5 and $8 in autoradiographs of the one-dlmensional gel slabs in which reaction products were analysed. Degradation products of this type, and any smaller fragments which migrated out of the gels during electrophoresis, were not recovered since only gel zones containing uncleared complex G and released proteins $5 and $8 were eluted. However, it is not clear why protein degradation should be confined to the initial phase (0 to 8 h) of the ammonolysis reaction.

(2) The ammonolysis reaction does not proceed to completion. Cleavage products (proteins $5 and $8) account for a maYimum of 60% of the total radioactivity recovered in identifiable form from ammonolysis reaction mixtures in the experiment described in l~igure 3. The remaining 40~/0 is recovered as unreacted complex O. These results arc obtained after reaction for 16 hours and do not change if the dura- tion of ammonolysis is increased to 24 hours. The effective yield of the overall process (ammonolysis followed by one-dimensional gel clectrophoresis) calculated as the ratio of the radioactivity recovered in $5 and $8 to tha t of the sample of complex G used for ammonolysis is therefore about 24°/o. For the identification of ammonolysis products, reaction mixtures arc analysed by two-dimensional polyacrylamide gel elcctrophoresis and losses of material encountered during this process are greater than those observed during the one-dimensional analyses used to obtain the data shown in Figure 3. l~Ieasurement of the effective overall yield after two-dimensional analysis of the products of ammonolysis of complex G gives values in the range 12 to 18%. The successful identification of the components of cross-Hn~ed complexes therefore requires the presence of relatively large amounts of radioactivity in samples of cross-linked complexes used for ammonolysis. For this reason many new species

ethanolamine .HC1 (pH 7.2), 10 mM-MgC]2, 0.05 M-KC1. The pH of the solution was then raised to 8-2 by addition of 2 M-triethanolamine, 10 m~-MgCl2, 0.05 M-KC1; 0-1 vol. of a solution of DMS (Materials and Methods) was added and the mixture was incubated at 80°C.

Samples of the reaction products (50 ~1) removed at time zero (hnmediately after addition of DMS) and at subsequent 80-rain intervals were diluted 100-fold with 0.01 ~-triethanolamine. HC1 (pH 7.2), 0.1 mM-Mg-acetate and filtered through nitrocellulose membranes whichwere thenwashed with 5 ml of the same buffer, dried and counted in 2 ml of scintillation fluid. The assay background of N 100 ets/min (time zero sample) was subtracted from all experimental values. The Figure shows the variation of DMS-dependent membrane-bound radioactivity ([SH]Lys-tRNA attached covalently to 70 S ribosomes) as a function of time of incubation with DMS. (25% of the total ribosome bound [SH]Lys-tRl~iA becomes covalently linked by DMS treatment.)

'_o

0-4

Q: 0.2


\ 0.6 -', , - -

~ & ~ . . . . . . . . • . . . . . . •

l-5

o / ' O • o o o @

• - I

0.5

I I I I 11 I 2 4 6 15 2 4

I I 8 I 0

Time (h |

u

c~9

" v

609

FIG. 3. Kinetics of ammonolysis of a binary complex containing proteins $5 and $8 (complex G. see Fig. 5 and Results, sections (e} and (d)}.

~sS-labelled 30 S ribosome subunits were treated with DMS and their proteins were fractionated in a 2.dimensional polyacrylamide gel. The radioactive material eluted from the spot designated G (Fig. 5(c)} was subjected to ammonolysls as described in Materials and Methods.

After incubation for the indicated times cleavage products and unreacted complex were precipitated from portions of the reaction mixtures as described in Materials and Methods. Precipitated material which contained ~ 90~o of the input radioactivity at all reaction times was analysed by elec~rophoresis in one dimension in 18~o polyacrylamide gel slabs in the presence of sodium dodecyl sulphate. After clectrophoresis gel slabs were dried and autoradiographed, zones containing complex G and proteins $5 and $8 were cut out and eluted, and recovered radioactivity was measured.

- - O - - O - - , Sum of the radioactivities recovered in eluates of gel zones containing released proteins $5 and $8 expressed as a fraction of the total radioactivity eluted from gel zones containing $5, $8 and unchanged complex G. (Yield of released proteins $5 -{- $8, left-hand scale.}

- - • - - • - - , Sum of the radioactivities recovered in eluates of gel zones containing unchanged complex G and released proteins $5 and $8 expressed as a fraction of the total amount of radioactivity (complex G) submitted to ammonolysis. (Yield of identifiable ammonolysls products, left-hand scale.)

Ratio of the radioactivity recovered in $5 to that recovered in $8 (O)- (Right-hand scale. The arrow indicates the theoretical ratio for an equimolar mixture of $5, and $8 (5, and 4 S atoms per molecule, respectively, see Table 1).)

present i n small amoun t s i n the pro te in complements of b is imidoester- t reated ribosomes a n d which p robab ly correspond to cross-linked complexes have no t been

inves t iga ted (e.g. several u n n a m e d , fa in t ly s ta ined spots visible in the upper r igh t -hand

region of Fig. 5(c)). (3) The ra t io of radioact iv i t ies recovered in prote ins $5 a nd $8 is approx imate ly

1"25 (Fig. 3), as expected for a b i n a r y complex since $5 a nd $8 conta in five a n d four

su lphur a toms, respectively. Since this ra t io shows no significant t ime-dependen t va r i a t ion t h roughou t a 24-hour ammonolys is t r ea tmen t , i t can be concluded t h a t a n y

processes leading to loss of radioact ive mater ia l dur ing this reac t ion are non-select ive

wi th respect to these two prote ins a n d therefore do no t interfere wi th the calculat ion

of the s to ich iometry of cross-]inked complexes.

610 A. E X P E R T - B E Z A N ( ~ O N E T A L .

(b) Reaction of bisimidoesters with 30 S ribosome subunits

(i) Absence of formation of dimers of 30 S particles

30 S ribosome subunits isolated in the presence of 0.1 mM-Mg 2+ dimerize in media containing 10 mM-Mg 2+ and dimers are readily detectable by sedimentation of 30 S subunits of this type in sucrose gradients containing high concentrations of Mg 2+ (Gualerzi & Cammarano, 1969; Gualerzi et al., 1973). In contrast, 30 S subunits prepared in the presence of 10 mM-Mg 2+ as described in Materials and Methods do not dimerize detectably under these conditions (GuSrin & Hayes, unpublished results). However this observation does not exclude the possibility that 30 S subunits prepared as described here form dimers which dissociate too readily to be detectable in sucrose gradients. Treatment of 30 S subunits with bisimidoesters is carried out in the presence of 10 m~-Mg 2+ and could therefore lead to the formation of stable 30 S dimers by the introduction of covalent cross-links between members of unstable binary complexes. The results presented in Figure 4 show that this does not take

x ._c E

:r.

5 D

4

3

2 •

I

Io

\

I I 20 Top

Fraction no.

FIG. 4. Sedimentation properties of DMS-treated 30 S ribosome subunits. Samples of control and DMS-treated (Materials and Methods) 3sS-labeUed 30 S subunits containing 4 × 104 cts/min of zsS were analysed by centrLfugation for 16-5 h at 18,000 revs]min at 4°C, on 4"8 ml, 5% to 20% sucrose gradients prepared in 0.01 M-Tris.HC1 (pH 7-4), 0.4 M-NaC1, 0.01 ~-MgC12, 6 mM-fl- mercaptoethanol using 3 place adapters in the Spineo SW25 rotor (Gu6rin & Hayes, 1973). After eentrifugation gradients were collected in 30 fractions, whose radioactivity was measured. In the Figure, the 2 sedimentation profiles obtained have been superimposed. - - O - - Q - - , DMS-treated 30 S ribosomes; ( . . . . . . ) control 30 S ribosomes.

The arrow indicates the position to which 50 S ribosomes sediment under the same conditions (30 S dimers obtained from 80 S subunits prepared in the presence of 0.1 mM-Mg 2+ sediment at 50 S). Results similar to those shown in the Figure were obtained when dimethyl succinhnidate and dlmethyl adipimidate-treated 30 S subunits were analysed in the same way bu t in these 2 eases no component sedimenting at 50 S was detected in the sedimentat ion profiles of the cross- linked 30 S subunite.

NEIGHBOURING RIBOSOMAL PROTEINS 611

place to a significant extent. However the proportion of dimers produced during DMS treatment of 30 S subunits prepared as described in Materials and Methods, though very low (HI O/o), is of the same order of magnitude as the yields of many of the cross-linked complexes analysed in the present report. To determine whether this small fraction of dimers contributes significantly to the population of cross-linked complexes which they contain, proteins isolated from an unfractionated preparation of DMS-treated 30 S subunits, and from the monomer fraction purified from this preparation by sucrose gradient sedimentation as shown in Figure 4, were compared by two-dimensional gel eleetrophoresis. The results of these analyses (not shown) were identical. All the new components present in the protein complement of unfractionated DMS-treated 30 S subunits (Fig. 5(c)) were present in comparable amounts in the proteins of monomeric DMS-treated 30 S particles.

(ii) Separation of cross-linked complexes

Figure 5 shows the results obtained when total protein isolated from 30 S ribosomal subunits treated with dimethyl adipimidate, dimethyl succinimidate, or DMS is analysed by the two-dimensional electrophoresis procedure of Kaltschmidt & Wittmann (1970) modified as described in Materials and Methods. Analysis of proteins isolated from 30 S ribosomes treated with dimethyl sebacimidate shows that use of this reagent yields no new complexes in addition to those formed by the action of DMS (results not presented). For this reason and because of its inconveniently low solubility in aqueous media the products of the action of dimethyl sebacimidate on ribosomal subunits have not been further investigated. Comparison of the autoradiographs (non-cross-linked control radioactive 30 S proteins) and photographs (Coo- massie blue-stained, bisimidoester-treated, non-radioactive proteins) of the same gel slabs in Figure 5 shows that the electrophoretic migration of ribosomal proteins which have been treated with bisimidoesters is modified in several respects. New protein species appear, particularly in the upper regions of gel slabs, which are occupied by material of low second dimension electrophoretie mobility. The electropboretic properties of these species suggest that they 'are complexes containing two or more ribosomal proteins joined by bisimidate cross-links. Their number rises as the span of the bisimidoesters increases. In addition it can be seen that stained spots (bisimidoester-treated proteins), irrespective of their positions in the gels, are more diffuse than neighbouring radioactive spots (control proteins).

We attribute the spreading of spots in two-dimensional gels of bisimidoester-treated ribosomal proteins to variations in the l~ind and degree of substitution produced by the action of these reagents, as exemplified in the following scheme in which reactions at a pair of neighbouring side-chain amino groups in a protein are considered. The choice of this example in which an internal cross-linl~ can be produced is justified by the fact that clustering of lysine residues is a characteristic feature of the structure of ribosomal proteins (WittmaIm & Wittmann-Li~bold, 1974).

Because the amidine group is more basic than the amino group which it replaces (Shriner & Neumann, 1944; Sober et al., 1970a,b), the intramoleeular substitution processes shown in this scheme will lead to conversion of ribosomal proteins to families of related species with different isoelectric points all more basic than that of the parent protein. In certain cases the existence of such a family of products is evident: for example, three products with electrophoretic mobilities very similar to that of protein $8 and the unchanged form of this protein form a group of close but resolved

+ NH

~ fJ

/OCI

-'I3

/NH-

-C--

(CHs

).--

C~

:Protein<

"~ N

H~

\NH

s

form

atio

n

+ 2

Protem(N*H-- (CH,

l.

\NH-

-C/

+

+ N

H2

v {

~OCHs

) Protein< NH-~

(CH2

)n--

C<0

\NH

2

/NH

s C

Hs0

\

/0C

Hs

Protein<

+ +

/~C--(CHs)n--Cxx

~ +

\NH

~

. .

H2

N+

~/

NH

s

. H

20

+ NH

2 U

/0C

H~

/N

H--

C--

(CH

2)~

--C

~

C NH-

-C--

ICH2

)n--

C%

N~H

2 0

+

Rea

ctio

n w

ith

side

- ch

ain

amin

o gr

oups

in

othe

r ri

boso

mal

pro

tein

s

Inte

rmol

ecul

ar c

ross

-lin

ked

com

plex

es

NEIGHBOURING RIBOSOMAL PROTEINS 613

spots in the sts.ln~ng pattern of polyacrylamide gels upon which proteins of dimethyl adepimidate-treated 30 S ribosomes are analysed (3 spots to the right of $8 in Fig. 5(b)). Elution and ammonolysis of the three $8 satellite spots shows that each con- rains only $8.

The overall effect of treatment of ribosomal proteins with bislmldoesters is therefore expected to be:

(a) the production of complexes joined by intermolecular eross-Hnl~s and

(b) the conversion of non-cross-Hnl~ed proteins to famflles of closely related derivatives which will appear in two-dlmensional gels as diffuse spots dis- placed, with respect to the parent proteins, towards the cathode o£ the first dimension of electrophoresis. This displacement is apparent in (b) (dimethyl adipimidate) and (c) (])MS) of Fig. 5. I t has, however, been observed repro- ducibly that proteins isolated from ribosomes treated with dimethyl succinimidate show a displacement in the opposite direction as is seen in (a) of Figure 5. A possible explanation for this effect is suggested by the observation (Protiva et al., 1950) that treatment of ethyl-~-carbethoxypropionlml- date(I) with ammonia under mild conditions leads to formation of 5-1mlno-2- pyrrolidone(III) presumably via ~-carbethoxypropionamidine(II).

CH=--CH2 I CH2--CH2 1 ["'"-7 I I N t t 3 i n / I I /

CzHs0--C C--0C2Hs ~ ~IH2--C C--0C2Hsl -* HN 0-FC=Hs0H . e t h a n o l JJ Jl at room J JJ ]1 J

NH O t empera tu re [ N H O j H

(I) ( I I ) ( I I I ) H-C_~HsOH

Non-cross-linked proteins present in the products of reaction of ribosomes with dimethyl succinimidate will contain only, or almost only, reagent molecules which have reacted at one end with a protein side-chain amino group and at the other with a water molecule (the presence of occasional internal cross-Hnl~a cannot be excluded as noted above). When the structure of these substituent groups is written as shown below, their analogy to the postulated amidine intermediate (II) becomes apparent and suggests that they may cyclize to im~nopyrrolidone residues. The occurrence of such a structural rearrangement during the treatment of ribosomes with dlmethyl succinlmidate would cause replacement of basic side-chain amino groups in proteins by less basic iminopyrrolidone residues with consequent reduction of the isoelectric pH of the substituted proteins, and therefore a displacement towards the cathode in the first dimension of polyacrylamide gel electrophoresis.

Cyclization processes of this type are not expected to occur during reaction with dimethyl adipimidate or ])MS since in the case of these reagents they would lead to the formation of seven and nine-membered rings, respectively. An arbitrary nomen- clature (see Fig. 5) is used to identify additional species present in the protein complement of bisimidoester-treated 30 S ribosomal subunits, and also a number of species whose electrophoretic behaviour corresponds to that of unmodified ribosomal proteins but whose apparent molecular weights are too high (low second dimension electrophoretic mobility) to exclude the presence of complexes. The protein contents of spots identified in this way in (a), (b) and (c) of Figure 5 have been characterized.

614 A. E X P E R T - B E Z A N T O N E T .4/; .

FIo. 5. Two-dimensional electrophoretic analysis of control and bisimidoester cross-linked S0 S ribosomal proteins.

Mixtures containing 200 ftg of proteins isolated from non-radioactive S0 S subunite treated with dimethyl succinimidate, dimethyl adipimidate, and DMS and 2 to 3 pg (10 e ors/rain of asS) of protein prepared from 3sS-labelled control 30 S subunits were analysed by 2-dimensional electrophoresis as described in Materials and Methods. In each panel of the Figure, the positions of


Protein--NH2 -!- CH2--CH2 ÷ H20

] IC__OCH3 CHsOC

615

CH2--CH2 I I

Protein--NH--C C~0CH3 -[- 2CH~0H II I] NI-I 0

Pro tc in - -N~ ( _ _ / f l ~--~0-{-CHaOH

" I N , " H

(c) Homogeneity and molecular weight of cross-linked complexes Figure 6 summarizes the results ob ta ined b y sodium dodecyl sulphate /polyacryl -

amide gel electrophoresis of rad ioac t ive mater ia l e luted from spots identif ied in Figure 5 as possible cross-l inked complexes. Of t e n spots whose eluates were ana lysed

J

radioactive spots (ssS-labelled control proteinst) detected by autoradiography of a dried gel slab have been drawn onto a photograph showing the positions of stained spots (bisimidoester-treated droteins) in the same gel.

(a) Dimethyl suceinimidate-treated 30 S subunits; (b) dimethyl adipimidate-treated 30 S subunite; (0) DMS-treated 30 S subunits.

t I t will be noted that 30 S protein S l l is not shown in Pigs 5 and 7 and is not represented in the histograms in Fig. 8. S11 is found in very small amounts in E. coZi 30 S subunits isolated either in the presence of 0.1 mM-Mg 2+ or 10 m~-Mg 2., 0-4 M-Na + because much of it remains with the 50 S subunit when 70 S ribosomes are dissociated (Morrison et aZ., 1973). Its presence is usually indicated by a dotted outline in photographs of 2-dirnensional gels (e.g. Kaltschmidt & Wittmann, 1970). When small samples of 35S-labelled proteins (200 to 300 ~g, 10 s ors/rain of asS; see Materials and Methods) are analysed by 2-dimensional gel electrophoresis as described here, S l l is not detectable in stained gels or in autoradiographs prepared from them.

616 A. EXPERT-BEZAN(~ON E T AL.

in this way, six (B, 02, D1, F1, L, J) contained a single radioactive species, two (C1, G) contained a single major component together with small amounts of con- taminants, and two (I, F2) contained two major components. The molecular weights of the eluted complexes calculated by reference to haemoglobin monomer and oli- gomer standards are shown in the last column of Table 2, and as expected, all fall in the range 25,000 to 60,000.

(d) Protein composition of cross-linlced c, ora~lexes

Three examples typical of the results obtained by two-dimensional electrophoretic analyses of the products of ammonolysis of cross-linked complexes are shown in Figure 7. I t may be noted tha t radioactive spots are usually more diffuse and larger than the stained spots of control proteins. This difference may be due to incomplete removal of bisimldoester residues bound at only one end to protein amino groups. As discussed in the ease of the preliminary analysis of cross-llnked proteins (section (b)) incomplete removal of these residues will yield a family of products with differing eleetrophoretie mobilities. In some cases the diffuseness of radioactive spots containing cleavage products complicates their identification, e.g. differentiation of proteins S15, S16, S17 which are located close to each other in two-dimensional gels is difficult.

To estimate the relative amounts of ammonolysis cleavage products detected in two-dlmensional gel slabs such as those shown in Figure 7, all stained and radioactive spots are cut out and their radioactivity is measured. Figure 8 contains the results of these measurements, presented in the form of histograms, for the materials eluted from 12 radioactive spots identified by letter in Figure 5. In these histograms released proteins are identified by number and their sulphur content is indicated

Fig. 6. Homogeneity and molecular weight of DMS cross-linked complexes isolated from bisimidioester-treated S0 S ribosomal subunits.

Samples of materials eluted from spots B, C1, C2, G, I, D, F1, F2, L and J (Fig. 5(c)) were subjected to eleetrophoresis in a vertical polyacrylamide gel slab in the presence of sodium dodeeyl sulphate according to Lacmmli (1970). A mixture of haemoglobin oligomers (Hb2, libS, Hb4, Mr = approx. 81,000, 46,500, 62,000, respectively) analysed in sample slots at each side of the gel slab provided visible molecular weight standards, whose positions are indicated in the photograph. Materials eluted from spots with the same designation in Ca), (b) and (c) of Fig. 5 gave the same results when analysed in this way (see text).


FIG. 7. Two-dimensional elec~rophoretic analysis o£ the products of A~mmonolysis of complexes F1, L and D1 (see Fig. 5).

Ammonolysis products of mixtures containing 3sS-labelled materlal (50,000 cts/rn~u) e|u~ed from spots F, L and D1 (Fig. 5(c)) and excess unlabeUed total 30 S protein were analysed by 2-dimensional electrophoresis. Gel slabs were stained, dried and autoradiographed (exposure time 10 days, see Materials and Methods for details). In each panel of the Figure the positions of control


(atoms per molecule, numbers in parentheses). This value, upon which the estimation of the stoichiometry of cross-lln~ed complexes is based, is known with precision only in the case of proteins $4, $6, $8, $9 and $18. In all other cases it is deduced from published measurements of the methionine and cysteine contents, and molecular weights of ribosomal proteins (see Table 1). Possible errors in these measurements limit the precision with which the sulphur content of proteins can be estimated and we consider the values given in the last column of Table 1 as probably correct to within ± 1 sulphur atom. The radioactivity per sulphur atom in the various ammono- ]ysis products identified in Figure 8(a) and (b) calculated from their measured total radioactivity and estimated sulphur content (Table 1) is listed in column 6 of Table 2. I t can be seen tha t in most cases approximately the same amount of radioactivity per sulphur atom is found in each component of a complex as expected if labelling of ribosomal proteins by 8sS is uniform, and if only one molecule of each protein is present per molecule of complex. Columns 8 and 9 list the calculated molecular weights of the various complexes (sum of the published molecular weights of identified cleavage products) and the molecular weight values estimated by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of the complexes (see section (c)). The following comments are relevant to the interpretation of the data shown in Figure 8 and in column~ 6 to 9 of Table 2.

(1) The amount of radioactivity found in protein $8 (complexes G, B, C1) is usually too small relative to the amount found in the other components of a complex containing this protein. Low recovery of $8 is an artefact of the modified two-dimensional gel electrophoresis procedure used in our experiments. The isoelectric point of $8 is very close to the pH of first-dimension electrophoresis, and examination of published

30 S proteins (stained spots) in a stained and dried gel slab have been drawn onto a photograph of an autoradiograph of the same gel slab.

(a), (b) and (c) The results of analyses of the products of ammonolysis of complexes F, L and D 1, respectively.

(N.B. The intensity of spots in autoradiographs of dried gel slabs is not directly proportional to their content of 35S.)


TABLE 1

Sulphur content of proteins from 30 S ribosomal subunits

619

Molecular weight Molar content Molar content of cysteinc Protein (X 10 -3) ofmethionine (a), (b) (c) (d)

Total sulphur content

(atoms/molecule)

S1 65 10 1.26 1-75 1 11 $2 28.3 5.8 0.88 0.74 1 6 $3 28.2 3.7 0-9 4-5 $4 26.7(22.5) 2.2(3) 1 (1) 0-79 0.85 4 $5 19.6 3.6 4 $6 15.6(16.5) 5-4(7) 7 $7 22.7 3.3 3 $8 15.5(14.2) 4.4(4) 0.88(1) 0.98 1 5 $9 16.2(14.5) 2.1(3) (0) + S l l ---- 0.9 3 S10 12.4 1.8 2 S l l 2.1 1-73 +$9 ~ 0.9 2 S12 17.2 0.5 3-4 2-69 4 3-4 S13 14.9 1.6 1 0-95 1 2-3 S14 14 2.26 1 0-97 0.85 3 S15 12.5 1.44 0.8 t 1 S16 11.7 1 0 1 S17 10.9 2 1.74 2 S18 12-2(8.95) 0(0) 1(1) 1.08 1.1 1 S19 13.1 2.2 2 $20 12 2.7 3 $21 12.2 0 0.6 1.04 2.15 1-2

Sulphur content of 30 S ribosomal proteins (atoms/molecule) deduced from published amino acid analysis. Data listed are taken from the following publications.

Molecular weights: Dzionara et al. (1970). Methionine content: Kaltsehmidt et at. (1970). Cystcine content: (a) Acharya & Moore (1973);

(b) Moore (1975); (c) Kahan et al. (1974); (d) Bakardjieva & Criehton (1974).

Figures in parentheses are derived from the results Of amino acid sequence determinations: $4, Reinbolt & Schiltz (1973); $6, Hitz et at. (1975); $8, Stadler (1974); $9, Chen& Wittmann-

Liebold (1975); S18, Yaguchi (1975). t Uncertain, see footnote to Table 1 in Acharya & Moore (1973).

two-d imens iona l e lec t rophore t i c p a t t e r n s o f E. coli 30 S r ibosoma l p ro te ins o b t a i n e d b y t h e or ig inal p rocedure o f K a l t s c h m i d t & W i t t m a n n (1970; e.g. Fig . 1 in t h a t paper ) shows t h a t i t f r equen t l y r ema ins p a r t l y in t h e f i r s t -d imens ion gel s t a r t i n g zone. I n t he e lec t rophore t i c sy s t em used here ( B a r r i t a u l t et al., 19765) t h e f i rs t -d imen- s ion s t a r t i ng zone is n o t po lymer i zed a n d a n y p ro t e in r ema in ing in i t a t t he end of

t h e f i r s t -d imens ion e lec t rophores is is lost . (2) The sum of t he molecu la r weights o f t he p ro te ins ident i f ied in t h e ammono lys i s

p r o d u c t s o f complexes I a n d F 2 is cons ide rab ly g rea t e r in each case t h a n t h e molecu- l a r weights of these complexes m e a s u r e d b y sod ium dodecy l s u l p h a t e / p o l y a c r y l a m i d e gel e lec t rophores is (section (c), above) . I t has a l r e a d y been n o t e d t h a t these two complexes are b o t h reso lved in to two componen t s b y e lec t rophores is in t h e presence o f sod ium dodecy l su lpha t e (section (c)). The molecu la r weights of t h e two componen t s p r e sen t in spo t F 2 a n d of t h e t h ree p ro te ins ($7, $9, S13) l i be r a t ed b y the i r a mmono- lys is a re c o m p a t i b l e on ly wi th t h e presence of two b i n a r y complexes $7-S13 a n d

620 A. E X P E R T - B E Z A N ~ O N E T JIL.

$9-S13 in this spot. According to this conclusion, material eluted from spot 172 contains two moles of S13 per mole of $7 or $9 and the amount of radioactivity found per sulphur atom in S13 should therefore be divided by two (Table 2, column 6) before comparison with the corresponding values for $7 and $9.

~mmono lys i s of mater ia l eIuted from spot I l iberates pro te in $8 a n d one or more

of proteins S15, S16, S17 which, as a l ready ment ioned , are no t readi ly d is t inguishable

b y two-dimensional gel electrophoresis in the system used here. The two-d imens iona l

electrophoretie procedure described b y S u b r a m a n i a n (1974; second d imens ion

4- 'o 6 x .c E 5

(3

4

.> 3

B tY

T: 0 1 5 0 0

v

I000

500

C2

$5 (4)

- $ 4 ' 50

(3) i

20

7 0

$8 ._= - (5) E

o

"G

- SI5-16-17 ( I - 2 ) ->

~ .~., ~.~.

$5 (4)

4 0 -

- G $8 (5)

- I

$5 4 - (4)

3 - $8 (5)

2 - SI5-16-17

( I - 2 )

T V T T ~r -T

2 4 6 8 10121415 2 0 2 4 6 8 I0 EI41520 16 16 [7 17

30 S ribosomol proteins

la)

G B

4- B

CI

$2 $3 (6)(4-5)

2

246 8 I0121420 16 17

Fie. 8 (a) and (b) Distribution of radioactivity among the products of ammonolysis of DMS cross-linked complexes.

The products of ammonolysis of 3sS-labelled material eluted from spots identified by letter in Fig. 5, and presumed to contain cross-linked protein complexes, were isolated as described in Materials and Methods and analysed by two-dimensional electrophoresis in the presence of excess unlabelled normal 30 S proteins. After staining and autoradiography all stained spots and un- stained radioactive spots were cut out of the gel slabs and their content of radioactivity measured. The results obtained are presented as histograms. Where it is not indicated ( • ) the scintillation


electrophoresis in the presence of sodium dodecyl sulphate) provides better separation of S15, S16 and S17. Analysis of the ammonolysis products of spot I material in this way clearly identifies $8 and S15 and a third component which is either S16 or S17. We conclude that spot I contains complex $8-S15 and either complex $8-S16 or $8-S17. The molecular weights of the two components present in spot I measured by electrophoresis in the presence of sodium dodecyl sulphate are compatible with this interpretation (see Table 2).

(3) Complex J does not contain protein $2. The relatively large amount of radioactivity found at the position of this protein after analysis of the products of ammonolysis of complex J (l~ig. 8(b)) is due to their content of unchanged complex which possesses e]ectrophoretic properties very similar to those of $2.

$9(3) FI DI / L ' $ 7 n FI

(3) SI3

x

,o

~ SI9 (l)

./--

i I [ I

i i

x C

/

24 681012141520 24 6 81012141s20 24 6810 12141520 is ,s I~ 17 17

30 S ribosomal proteins (b) spect rometer background count has been deducted. I n all cases proteins S15, S16 and S17 which are no t well resolved in the 2-dimensional electrophoresis sys tem used were eluted and counted together . Released proteins considered to be present in significant amounts and, in the cases of complexes G, B and F1, residual amounts of unreac ted cross-linked complex are identified a t the top of the re levant bars in the histogram~. Figures in parentheses below these identifications are the su lphur contents of r ibosomal proteins (atoms/molecule, see Table 1).

TA

BL

E

2

Mol

ecul

ar w

eigh

ts a

nd p

rote

in c

ompo

sitio

ns o

f cro

ss-l

inke

d co

mpl

exes

isol

ated

from

dim

ethy

l su

beri

mid

ate-

trea

ted

30 S

rib

osom

al s

ubun

its

Ob

serv

ed

Mea

sure

d r

adio

acti

vit

y

Pro

po

sed

S

um

of

mo

l. w

t to

ol.

wt

of

Mol

. w

t N

um

ber

of

(ets

[10

rain

(e

ts]1

0 m

in p

er

com

po

nen

t co

mp

lex

C

om

ple

x

Pro

tein

co

mp

osi

tio

n

( ×

10

-3)

sulp

hu

r at

om

s ab

ov

e su

lph

ur

ato

m)

pro

tein

s (S

DS

-gel

b

ack

gro

un

d)

of

com

ple

x

( ×

10

- a)

el

ectr

op

ho

resi

s)

( × 1

0 -3

)

A

$2

28.3

6

6400

10

70

$2

-$3

56

.5

54

$3

28-2

4/

5 56

50

1130

1140

0

G2

$4

26.7

4

2700

67

5 $5

19

.6

4 52

00

1300

$

4-$

5

46.3

46

B

$4

26.7

4

1350

34

0 $

4--

$5

-$8

61

.8

59

$5

19.6

4

2000

50

0 $8

15

.5

5 20

00

400

G

$5

19.6

4

45,0

00

9000

$

5-$

8

35.1

34

$8

15

.5

5 22

,000

44

00

C1

$5

19.6

4

3400

85

0 $

5-$

8-S

15

47

.6

$8

15.5

5

1900

38

0 o

r $

5-$

8-S

16

46

-8

46

S15

/16/

17

12.5

]11.

7/10

.9

1]1/

2 70

0 35

0-70

0 o

r $

5-$

8-S

17

46

-0

I $8

15

-5

5 90

0 18

0 $

8-S

15

+

28

31

+ 2

6 S

15

12-5

1

300

300

$8-S

16 o

r 27

-2

S16

/17

11.7

/10.

9 1/

2 30

0 30

0/15

0 $8

-S17

26

.4

J $6

15

-6

7 12

,000

17

00

$6-S

18

27.7

28

S

18

12.1

1

1500

15

00

F1

$7

22.7

3

12,4

00

4100

$

7-$

9

28.9

35

$9

16

.2

3 10

,500

35

00

F2

$7

22

.7

3 90

00

3000

$7

-S13

37

.6

37

+3

3

$9

16.2

3

9000

30

00

+ S

13

14.9

2/

3 19

,500

65

00/9

800

$9--

S13

31

-1

I)l

$7

22.7

3

14,5

00

4800

S

13

14.9

2[

3 13

,800

46

0016

900

S19

13

.1

1/2

3500

17

50/3

500

$7

-81

3-8

19

50

.7

44

S13

14

-9

2/3

64,0

00

21.0

00/3

2,00

0 S

13-S

19

28.0

28

L

S

19

13.1

1/

2 26

,000

13

,000

/26,

000

Mol

ecul

ar w

eigh

ts l

iste

d in

co

lum

n 9

are

ave

rage

val

ues

calc

ulat

ed f

rom

th

e re

sult

s of

2 o

r m

ore

elec

trop

hore

tie

anal

yses

of

the

typ

e sh

own

in F

ig.

6. T

he

prec

isio

n of

the

se e

stim

ates

is

lim

ited

(ap

prox

. 10

00)

by

th

e b

road

nes

s of

ban

ds

of ]

DM

S-g

ener

ated

hem

og

lob

in o

ligo

mer

s. D

ata

in c

olum

ns 5

an

d 6

wer

e o

bta

ined

fro

m e

xp

erim

ents

des

crib

ed i

n R

esul

ts,

sect

ion

(c)

and

(d)

. T

he

valu

es g

iven

in

colu

mns

3 a

nd

4 a

nd

th

e ca

lcul

ated

mol

ecul

ar w

eigh

ts o

f co

mpl

exes

in

col

umn

3 ar

e de

rive

d fr

om

pu

bli

shed

res

ults

whi

ch a

re s

um

mar

ized

in

Tab

le 1

. S

DS

, so

dium

dod

ecyl

sul

phat

e.

624 A. E X P E R T - B E Z A N Q O N E T A L .

(4) Radioact iv i ty found a t the position of protein 87 after analysis of the ammonolysis products of complex L is due to contaminat ion by residual unreacted complex.

(5) Analysis of the ammonolysis products of material eluted f rom spots M and N in the two-dimensional gels of proteins of DMS-treated 30 8 ribosomes (Fig. 5(e)) showed tha t neither contained a cross-linked complex. Spot M contains only protein 85 and spot N only protein 84, the lat ter result providing a striking demonstrat ion of the alteration of the electrophoretie mobili ty of a protein by intramolecnlar sub- st i tution (Fig. 7(c)).

(6) The products of ammonolysis of complex D1 do not contain detectable amounts of this complex, which migrates more slowly in the second dimensions of electrophoresis than 30 S protein 83 (see Fig. 5(0)), The radioactive spot si tuated to the right of and below $3 and $4 in Figure 7(c) displays eleetrophoretic properties similar to those of complex L (S13-S19) (see Fig. 5(a), (b) and (c)) and m a y in fact correspond to this complex which could be produced as an intermediate during ammonolysis of complex D1 ($7-S13-SI9).

4. D i s c u s s i o n

(a) Quantitative aspects of the cross-linking reaction of bisimidoesters Inspection of Figure 5 suggests tha t few of the large number of cross-linked protein

complexes detectable in the products of the t rea tment of 30 S ribosome subunits by bisimidoesters are formed in significant amounts. This conclusion is confirmed by the da ta shown in Table 3 which lists the yields of ten of the 13 complexes whose

TABLE 3

Yields of formation of bisimidoester cross-linlced complexes

Cross-linked complex

Yield obtained by treatment of 30 S ribosome subunits with bisimidoesters

Dimethyl suberimidate Dimethyl adipimidate

S13-S19 53 $5-$8 46 $6-818 25 $7-$9 23 $4-$5-$8 3 S8-S15 + S8-S16/17 2-3 $5-$8-S15]16]17 2 $5-$8-S15]16]17 2 $4-$5 2 $2-$3 ~ 1 $7-S13-S19 ~ 1

26 33 10 11

Complexes formed in trace amounts only by reaction with dimethyl adipimidate

Complex not formed by reaction with dimethyl adipimidate

Yields are calculated as follows. All radioactive spots are cut out of a gel slab such as those whose autoradiographs are shown in Fig. 5, and their radioactivity measured. The total amount of radioactivity present in proteins involved in the cross-linked complexes listed in the Table is calculated as the sum of the amounts found in these proteins in the free state and in complexes (e.g. total radioactivity in $5 -~ radioactivity in free $5 plus calculated amounts in complexes $5-$8, $4-$5-$8, $4-$5, etc.). The yield of each complex is the ratio of the total radioactivities found in the complex and in the proteins which it contains (yield of $5-$8 = total cts]min in SS-SSltotal ets]min in all forms of 85 ÷ total ors[rain in all forms of $8).

N E I G H B O U R I N G RIBOSOMAL P R O T E I N S 625

compositions have been determined, and shows tha t only four, $5-$8, $7-$9, $6-S18 and S13-S19, are formed in high yield (see the legend to Table 3 for details of the calculation of the yields). P rox imi ty of these protein pairs or interaction between their members has been inferred from other studies of 30 S ribosome structure. The assembly map of Nomura & Held (1974) shows the presence of strong interactions deduced from the results of reconstitution experiments between proteins $5 and $8, $7 and $9, and $6 and S18. Isolation of numerous ribonucleoprotein fragments from ribonuclease-digested 30 S particles further confirms the proYimlty of t h e

following protein pairs: $5, $8 found with other proteins in five fragments; $7, $9 in four fragments; S13, S19 in two fragments; $6, S18 in one fragment (Morgan & Brimacombe, 1972,1973).

Almost all the cross-]inl~ed complexes formed by the action of dimethyl suberimidate including the four produced in high yield are also formed, the lat ter in significant amounts (Table 3), when the shorter reagent dimethyl adipimidate is used and even, in two cases, by t rea tment with dimethyl succi~imldate, the shortest reagent tested (Table 4). Making the assumption tha t the cross-links established between the same proteins by different bisimidoesters involve the same pair o f - - N H 2 groups in each case, i t is possible to deduce approximate upper and lower limits for the distance between these groups from the da ta given in column 3, Table 4 and the known dimensions of the bisimidoesters. The values obtained are listed in the last column of Table 4. They show tha t the separation of bridged amino groups is approximate ly the same in complexes formed in high and in low yield, al though the two complexes

TABLE 4

Approximate distances between bridged amino grouts in bisimidoester cross-linked complexes of 30 S ribosomal proteins

Complex Composition

Presence in the products of treatment of 30 S ribosome subunits with bisimidoesters

DMSuc DMA DMS

Distance (d) between cross- linked amino groups (A)

G $5-S18

L S13-S19

+ ~- + d < 3.78

C2 $4-$5 B 84-$5-$8 I $8-S15 ~- $8-S16/17 C1 $5-$8-S15/16/17 A $2-$3 F1 $7-$9 J $6-S18

~- -p 3.78 < d < 8-8

F2 $7-S13 -p $9-S13 D1 $7-S13-S19 - - -- -~ 6.3 ~ d ~ 8.8

DMSuc, dimethyl succlnlm~date; DMA, dimethyl adipimidate. The values given, based on the known dimensions of bisimidoesters, are to be considered as

indicating the probable approximate limits of approach of the amino groups involved in cross-link formation.

{}26 A. E X P E R T - B E Z A I ~ O I ~ E T A L .

in which this distance has the smallest lower limit, $5-$8 and S13-S19, are formed in highest yield. This suggests that factors other than the distance between side-chain amino groups in neighbouring proteins influence the efficiency with which cross-links are introduced, e.g. accessibility of the region containing these groups to reagent molecules, competition by reaction of imidoester groups with water if the amino groups are situated in a hydrophilic environment, the presence of other amino groups in either or both proteins of a cross-linkable pair at distances such that intramolecu- lar bridges can be formed with consequent loss of one or both amino groups between which cross-llnl~s can be established, etc. Complete characterization of cross-linl~ed complexes requires determination not only of their protein compositions but also of the number and position of the cross-links between them. Data in Table 3 strongly suggest that information of the latter type will be accessible only in the case of complexes $5-$8 and S13-S19 and possibly $7-$9 and $6-S18.

(b) E~ciency of ammonolytic cleavage of bisimidoester cross-linlcs

Although the efficiency of ammonolytic cleavage of amidine cross-links under the reaction conditions described here is low, it appears to be considerably higher than that observed by other workers (Bickle et al., 1972). We believe that the higher yields of fission products which we observe are due to the increased solubility of cross-linked protein complexes produced by the presence of 2% (w/v) sodium dodecyl sulphate in ammonolysis reaction mixtures.

(c) Interpretation of results

The possibility that structural alterations are induced in ribosome subunits during reaction with bisimidoesters and that protein-protein proximity relations derived by the use of these reagents do not correspond to the native conformation of the ribosome must be considered. However, DMS-treated 70 S ribosomes in which the 30 S and 50 S subunits are covalently linl~ed retain 80% of the polyphenylalanine synthe- sizing activity of unmodified ribosomes (Slobin, 1972), and active 30 S subunits can be reconstituted in vitro from 16 S ribosomal RNA and a protein mixture containing a dimethyl adipimidate cross-linked complex of 30 S proteins $5 and $8 in place of the two free proteins (Lutter & Kurland, 1973) or the cross-linked complex S13-S19 in place of the latter two proteins (Lutter, 1973). These results show that reaction with bisimidoesters apparently does not cause significant changes in ribosome conformation, and therefore lead to the conclusion that these reagents can be used to identify neighbouring proteins in the ribosome.

Of the thirteen cross-Hnl(ed complexes analysed in this report (Table 2) eight ($4-$5, $5-$8, $4-$5-$8, $8-S15, $8-S16/17, $5-$7-S15/16/17, $7-$9, and $7- S13-S19) were described briefly in an earlier publication (Barritault et al., 1975a), four have been isolated and characterized by others (DMS cross-linked complexes $2-$3, $5-$8, $7-$9, S13-S19; Sun et al., 1974; Kurland, 1974), and one, $9-S13, has not been previously reported. Cross-linked complexes with the same protein compositions as most of those listed in Table 2 and a considerable number of additional complexes have also been obtained by the use of other bifunctional reagents (Table 5).

Several attempts have been made to deduce a structure for the 30 S ribosome from results of the type described here and data of a similar nature obtained by other techniques (Morgan & Brimacombe, 1973; Bollen et al., 1974; Traut et al., 1974;

N E I G H B O U R I N G RIBOSOMAL P R O T E I N S

TABLE 5

Protein-protein complexes identified in the products o f treatment o f E. coli 30 S ribosome subunits with cross-linking reagents

627

Cross-linking agent Complexes identified References

Dimethyl $5--$8, S13-S19 This work succinimidate

Dimethyl adipimidate

S2-S3, S4-S5, S5--S8, S6-S18, S7-S9, S8-S15, S8-S16]17, S13-S19, S4-S5-S8, $5-$8-S15/16/17

$5-$8,$7-$9, S13-S19

This work Barritault st al. (1975a)

Lutter et al. (1972) Bode et al. (1974) Lutter & Kurland (1973)

Dimethyl suberimidate

$2-$3, $4--$5, $5-$8, $6-S18, $7-$9, $8-S15, $8-S16/17, S13-S19, $4-$5-$8, $5-$8-S15/16/17, $7-S13-S19

$5-$8, $7-$9, S13-S19, S14-S19

$5-$9

This work Clegg & Hayes (1974) Barritault et al. (1975a)

Bode et al. (1974)

Bickle st al. (1972)

$2-$3, $2-$5, $2-$8, $3-S10, $4-$5, Lutter st aL (1974b,1975) Tartryl-diazide $5-$8, $6-S18, $7-$9, S13-S19

Tartryl-diglycylazide $4-$5 Lutter et al. (1974b,1975)

$2-$3, $5-$8 Sommer & Traut (1974a,b) Sun et al. (1974)

Sommer & Traut (1975)

Sommer & Traut (1976) Methyl-4-mercaptobutyrimidate

$2-$3, $5-$8, $5-$9, $7-$8, $7-$9

$2-$5, $2-$8, $3-$4, $3-$5, $3-$9, $3-S10, $3-S12, $4-$5, $4-$6, $4-$8, $4-$9, $4-S12, $4-S13, $4-S17, $5--S13, $6-S18, $7-S13, $8--Sll, $8-S13, Sll-S13, S12--S13, S12-$20, S12-$21, S13-17, S19-$21

Dimethyl-3,3" $2-$3, $4-$5, $5--$8, $4-$5-$8 Peretz et aL (1976) dithiobispropionimi- date

p-Phenylenebis- S18-$21 Chang & Flaks (1972) maleimide Lutter eta/. (1972)

Tetranitromethane Sll-S18-$21 Shih & Craven (1973)

Sommer & Traut, 1975,1976; Tischendorf et al., 1975). The results of all the cross- linking experiments carried out so far, including those described here, are compatible with the structure models proposed by these authors. However we believe tha t these models should be considered as purely speculative for the following reasons.


(1) As shown in Table 3, many cross-linked complexes whose formation is taken as evidence for the proximity of two or more ribosomal proteins are formed only in very small amounts. Preparations of purified 30 S ribosome subunits are known to be heterogeneous in protein composition and to contain a majority of functionally inactive particles. Cross-linked complexes formed in trace amounts could therefore be derived from modified particles with abnormal structures.

(2) The conformations of ribosomal proteins in the ribosome are unlmown. They are assumed to be spherical in all models so far suggested with the exception of tha t proposed by Sommer & Traut (1975,1976), which takes into account evidence that protein $4 has an elongated conformation in the isolated state (Garrett & Wittmann, 1973) and also in the intact 30 S subunit (Lake et al., 1974).

Financial support for this work was provided by the Centre National de la Recherche Seientifique (Equipe de Recherche no. 101), the Delegation k la Recherche Scientifique et Technique (A. C. C. no. 750199), the Commissariat k l'Energie Atomique, and the Fonds de la Recherche Mddecale Fran~aise.

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identification of neighbouring proteins in escherichia coli 30 s ribosome subunits

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