THE JOURNAL OF BIO~OGKAL CHEMISTRY Q 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265. No. 19. Issue of July 5. pp. 1133%11344,199O Printed in U.S. A.

Three-dimensional Localization of the NH2- and Carboxyl-terminal Domain of Ribosomal Protein Sl on the Surface of the 30 S Subunit from Escherichia coli*

(Received for publication, January 31,199O)

Jan Walleczekll , Renate Albrecht-Ehrlich, Georg StGfflerS, and Marina Stiiffler-MeilickeQ From the Max-Planck-Institut fiir Molekulare Genetik, Abt. Wittmann, Ihnestra(3e 73, D-1000 Berlin 33 (Dahlem), Federal Republic of Germany and the $Institut fiir Mikrobiologie, Medizinische Fakultlit, Universitcit Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria

Antibodies were raised against Escherichia coli ri- bosomal protein Sl and its NHZ- and COOH-terminal fragments, and their specificity was demonstrated by a variety of immunological techniques. These antibod- ies were then used to investigate the location of protein Sl and its NH2- and COOH-terminal domains on the surface of the 30 S ribosomal subunit by immunoelec- tron microscopy. In order to prevent dissociation of the protein during the experiments, Sl was cross- linked to 30 S subunits with dithiobis(succinimidyl- propionate); cross-linking yield was 100%. Epitopes of the NH*-terminal domain of Sl were localized at the large lobe of the 30 S ribosomal subunit, close to the one-third/two-thirds partition on the side which in the 70 S ribosome faces the cytoplasm. Experiments with monovalent Fab fragments specific for the COOH-ter- minal part of Sl provide evidence that the COOH- terminal domain forms an elongated structure extend- ing at least 10 nm from the large lobe of the small subunit into the cytoplasmic space.

Among the ribosomal proteins of Escherichia coli, protein Sl from the 30 S ribosomal subunit has some remarkable features: (i) with a molecular weight of 61,159 it is by far the largest rihosomal protein (Wittmann-Liebold, 1986). (ii) It is acidic, whereas most other ribosomal proteins are basic. (iii) Its association with the ribosome is weak and reversible, whereas most ribosomal proteins bind strongly to the ribo- some (Subramanian and van Duin, 1977; Laughrea and Moore, 1977). (iv) It is the only ribosomal protein with a high affinity for mRNA (e.g. Draper and von Hippel, 1978). Protein Sl is also an exception in that it is one of the few ribosomal proteins to which a definite functional role has been assigned; several research groups have established that protein Sl is essential for efficient translation of mRNA by the ribosome (e.g. Szer and Leffler, 1974; Van Dieijen et al., 1976; Sury- anarayana and Subramanian, 1983) and the loss of its func- tion by mutagenesis is lethal to the cell (Kitakawa and Isono, 1982).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Present address: Institut ftir Klinische und Experimentelle Virologie, Hindenburg- damm 27, D-1000 Berlin 45, Federal Republic of Germany.

II Present address: Bioelectromagnetics Research Facilitv, Re- search Medicine and Radiation Bioph;isics Division, Lawrence Berke- ley Laboratory, University of California, Berkeley, CA 94720.

Physical studies have revealed that in solution Sl is an elongated structure of approximately 23 nm in length (Laugh- rea and Moore, 1977; Giri and Subramanian, 1977; Yokota et al., 1977; Labischinski and Subramanian, 1979), which would imply that Sl is as long as the entire 30 S subunit. Further- more it has been shown by small-angle x-ray scattering (La- bischinski and Subramanian, 1979), H NMR (Moore and Laughrea, 1979), and fluorescence polarization measurements (Chu and Cantor, 1979) that Sl consists of two structurally distinct domains, results which are in agreement with the finding that mild trypsin digestion cleaves protein Sl into two fragments (Suryanarayana and Subramanian, 1979). Cleavage of Sl with cyanogen bromide yields three different fragments (Subramanian, 1983): The NH*-terminal fragment (Sl-F2a) contains amino acids 1-193, the middle fragment (Sl-F3) contains amino acids 224-309, and the COOH-ter- minal fragment (Sl-F2b) contains amino acids 332-547 (see Fig. 1). The NH,-terminal and COOH-terminal fragments exhibit different functional properties: the COOH-terminal domain of Sl strongly binds mRNA, whereas the NH&er- minal domain of Sl shows high affinity for the 30 S subunit, but not for mRNA (for a review see Subramanian, 1983).

The purpose of the work described here was to determine the locations of the NH,- and COOH-terminal domains of protein Sl on the three-dimensional model of the 30 S ribo- somal subunit by immunoelectron microscopy (IEM).’ In order to prevent dissociation of the protein during the exper- iments it was necessary to covalently attach protein Sl to the ribosome by chemical cross-linking. Whereas the NH,-ter- minal domain of protein Sl could be localized in a defined region on the ribosomal surface, the immunoelectron micro- scopic data obtained with monovalent Fab-fragments pro- vided evidence that the COOH-terminal domain of Sl is highly elongated in situ and extends from the ribosomal surface into the surrounding space.

EXPERIMENTAL PROCEDURES

Preparation of Ribosomes ana’ Ribosomal Protein(s)-70 S ribo- somal tight couples and 50 and 30 S ribosomal subunits from E. coli MRE 600 were isolated as described by No11 et al., (1973) with slight modifications). 30 S ribosomal subunits lacking ribosomal protein SI (30 S-Sl) were prepared from 30 S subunits as described by Suhra- manian (1983). Total ribosomal protein was extracted by the proce- dure of Leboy et al. (1964). Ribosomal proteins were analyzed on SDS polyacrylamide gels (Laemmli, 1970) by applying intact ribosomal subunits.

Protein Sl was isolated from 30 S subunits by affinity chromatog-

L The abbreviations used are: IEM, immunoelectron microscopy; SDS, sodium dodecyl sulfate; DSP, dithiobis(succinimidylpro- pionate); 30 S(-Sl), 30 S subunits that quantitatively lack protein Sl.

11338

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Localization of Ribosomal Protein Sl

FIG. 1. Linear representation of E. coli ribosomal protein Sl and its fragments. Rl-R4 represent the four internal repeats (after Suhramanian, 1983).

raphy over a poly(U) column (Subramanian, 1983). Fragments Sl- Wa, Sl-F3, and Sl-F2h were prepared from protein Sl by cleavage with cyanogen hromide and purified by chromatography over a DEAF-column (Subramanian et al., 1981).

l’rcpuratum und Characterization of Antlboclies-Antibodies spe- clfic for purified protein Sl were raised in sheep and antibodies against fragments Sl-F2a and Sl-F2b were raised in rabbits. The antlsera were characterized hy double immunodiffusion on cellulose acetate gels (Zuhke rt al, 1977) and immunoblotting (Breitenreuter c’t nl , 1984). IgG was prepared by first precipitating the immunoglob- uhns at 40% saturation with ammonium sulfate (pH 8.0). IgG from sheep was then puritied by DEAE chromatography (Stoffler-Meilicke cs/ a/, 1984). IgG from rabbit was prepared by affinity chromatography on protein A-Sepharose (Hjelm et al, 1972). Preparation of mono- valent Fah was according to Putnam et al. (1962) with some modifi- cations (Maschler, 1974).

Cros.vlrnhirtg of Protem SI to Ribosomes-Ribosomes were cross- linked with 1 mM DSP according to Lomant and Fairbanks (1976) with Some modifications. Typically, 500 AL,,~,,,,,, units of 30 S subunits were dialyzed against buffer A (50 mM triethanolammonium chloride (pII 8.(l), 50 mrvt potassium chloride, and 6 mM magnesium chloride) and subsequently DSP (5 mg DSP freshly dissolved in 150 ~1 of dimethyl sulfoxide) was added dropwise until the concentration of DSP reached 1 mM. The temperature was raised to 37 “C and gly- cmamide (1 M glycinamide in buffer A) was added to a final concen- tration of 50 mM m order to terminate the cross-linking reaction.

Separation of cross-linked monomeric ribosomes from ribosome dimers and multimers was achieved by sucrose gradient centrifuga- tion; approximately 150 AL ,,,,,,,, 1 ribosomes were layered onto a linear sucrose gradient (10-X0% sucrose in 100 mM ammonium chloride, 50 mM Tris-Cl (pH 7.4). 20 mM magnesium chloride, 5 mM glycinamide) and centrifuged in a Beckman SW27 rotor at 18.000 (70 S), 19.000 (50 S), and 23.000 rpm (30 S) for 16 h at 4 “C. The monomeric rihosome fractions were pooled and pelleted in a Beckman Ti75 rotor for 4 h at 4 “C. The ribosomes were resuspended in a buffer containing 60 mM ammonium chloride, 20 mM Tris-Cl (pH 7.4), and 10 mM magnesium acetate and stored at -80 “C.

Separatum of Ribosome-I& Complexes-Sucrose gradient centrif- ugation and preabsorption experiments were carried out as described previously (Stoffler-Meilicke et al., 1984). Monomer reduction (per- cent) was estimated by measuring the area under the 30 S subunit peak in the presence of IgG relative to that of the control subunits in the absence of IgG. Dimer formation (percent) was quantitated by determining the area under the dimer peak in the presence of IgG relative to the monomer peak of the control subunits.

Immuno&ctron Mlcroscops-Samples for electron microscopy were taken directly from the sucrose gradients and applied to grids using the double-layer carbon technique (Tischendorf et al., 1974). The specimens were contrasted with 2.5% uranyl formate. Electron microscopy was performed on a Philips EM 301 at an instrumental magnification of 110.000 at 80 kV accelerating voltage.

RESULTS

Characterization of the Antibodies-Antibodies were raised against intact protein Sl and its NH,- and COOH-terminal fragments and the antibodies were characterized by immu- noblotting. When the antisera were tested with total protein from 30 S subunits or 70 S ribosomes, they exclusively reacted with protein Sl and with no other ribosomal protein (Fig. 2, B-U, lane 4). When the antisera were tested for their reactiv- ity with the three different fragments, anti-S1 was found to react with all three of them (Fig. 2B). Fragment Sl-F3 was immunostained as two distinct bands (Fig. 2B, lane 5), with molecular weights of approximately 16,000 and 13,000. The

12 3

Sl --

s1-F20= - F2b

Sl-F3 -

A

11339

4 2 3 5 L 2 3 5 L235

-- _~. _- s-

t,. =

B c 0

FIG. 2. Characterization of the Sl-specific antibodies by immunoblotting. Lane I, 20 pmol of protein Sl: lane 2, 20 pmol of fragment Sl-F2a; lane 3, 20 pmol of fragment Sl-F2h; lane $, 20 pmol of 30 S subunits; lane 5, 20 pmol of Sl-FX A, one-dimensional SDS polyacrylamide gels. The proteins were stained with Commassie Brilliant Blue. R-0, immunohlots from one-dimensional SDS poly- acrylamide gels incubated with anti-S1 (R), anti-Sip>,, (0. and anti- Sh, (I)).

12 3 4 5

-_ - -s1

FIG. 3. One-dimensional SDS polyacrylamide gels. Lane 1, 30 pg of 30 S subunits; lane 2, 30 pg of 30 S subunits cross-linked with 1 mM DSP; lane 3, 30 pg of cross-linked 30 S subunits after cleavage of the cross-link with mercaptoethanole; lane 4, N pg of 30 S(-Sl) subunits; lane 5, 2 fig of ribosomal protein removed from 30 S subunits by poly(U) affinity chromatography (for details see text). For lanes l-3 a 15-25% gradient gel was used, and for lanes 1 and CT a 15% linear gel was used. The gels were stained with Coomassie Brilliant Blue.

M, of 16,000 is in agreement with the value published by Subramanian and co-workers (1981), whereas the band with the lower M, may represent a degraded form. This experiment also demonstrates that none of the Sl fragments was contam- inated with any other fragment.

Anti-SIFy, reacted exclusively with fragment Sl-F2a, whereas no reaction could be observed with fragment Sl-F2b nor with fragment Sl-F3 (Fig. 2C). Accordingly, anti-S1F9k, reacted exclusively with fragment Sl-F2b (Fig. 20). This immunoblotting analysis thus demonstrated convincingly that the fragment-specific antibodies reacted only with their cognate antigens.

Chemical Cross-linking of Protein Sl to 30 and 70 S Ribo- somes-In order to prevent dissociation, Sl was cross-linked to 30 S subunits, using cleavable DSP (cross-linking span 1.2 nm) as cross-linking reagent. Analysis of the proteins ex- tracted from cross-linked 30 S ribosomes by SDS gel electro- phoresis showed that the Sl band was missing from the gel, indicating that protein Sl had formed a high M,-weight complex with other ribosomal components which could not enter the separation gel (compare lanes 1 and 2 in Fig. 3). If, however, the cross-link was cleaved with mercaptoethanole, the Sl band reappeared on the gel, indicating that the high M, complex had been cleaved and that protein Sl could again migrate into the gel (see lanes 3 in Fig. 3). The fact that protein Sl was completely missing from the gel after cross- linking suggested that the extent of cross-linking was 100%.

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

11340 Localization of Ribosomal Protein Sl

Cross-linking of 70 S ribosomes gave the same results (data not shown).

Reactivity of Anti-Sl, Anti-Sin, and Anti-Slnb with Cross- linked 30 and 70 S Ribosomes-The availability of stable 30 S-IgG-30 S dimeric immunocomplexes is a necessary prereq- uisite for determining the location of an IgG binding site on the 30 S subunit by IEM. When any of our Sl-specific antibodies had been tested for reactivity with intact 30 S subunits or 70 S ribosomes by sucrose gradient centrifugation, none had yielded stable dimers, and we could demonstrate by immunoblotting that after reaction with Sl-specific antibod- ies the 30 S subunits (or 70 S ribosomes) lacked protein Sl, indicating that the protein had been removed from the ribo- some by the antibody (data not shown).

For this reason we had crosslinked protein Sl to 30 S ribosomes and when these cross-linked 30 S subunits were reacted with the SI-specific antibody, dimer formation was observed upon sucrose gradient centrifugation (Fig. 4B). With the Sl-specific antibody maximal dimer formation was seen at an IgG concentration of 0.1 A280 .,/l AzGO nm 30 S subunits (Fig. 4B). Dimer formation was very low, and since we had to take into account that in the 30 S control 2-10% of the ribosomes appeared as cross-linked 30 S-30 S dimers, even after several repurification cycles (see Fig. 4A, peak I) maxi- mal dimer formation was only approximately 6% (Table I). At higher antibody concentrations dimer formation ceased, as can be seen from Fig. 5A, where dimer formation (percent) is plotted against increasing anti-S1 concentrations. However, inspection of the gradient profiles obtained with high antibody concentrations revealed that a significant reduction of the monomer peak had taken place (Fig. 4C), indicating that the 30 S subunits had been precipitated by the antibody. When the amount of reduction of the monomer peak was taken as a measure for reactivity, up to 50% of the cross-linked 30 S subunits were reactive with the Sl-specific antibody (Fig. 5B). The fact that the 30 S subunits had been precipitated by the antibody was a first hint that protein Sl had several antigenic determinants exposed at the ribosomal surface, since precip-

B 1 2 1 I i

.:::

. Sedimentation

FIG. 4. Sucrose gradient profiles. 1 Azeonrn cross-linked 30 S subunits without antibody (A), “,,, incubated with 0.1 A2ao(B), and with 2 Azao,, anti-S1 IgG (peak 1 = 30 S dimers; peak 2 = 30 S monomers; peak 3 = IgG) (C).

TABLE I Reactivity of SZ-specific antibodies with 30 S subunits

and 70 S monosomes Maximal dimer formation and maximal monomer reduction are

given. They have been quantitated as described under “Experimental Procedures.”

Antibody \

Anti-S1 Anti-Sirs, Anti-Slrzb

Dimer formation

30 s 70 s

% 6 12 7 11 4 7

Monomer reduction

30 s 70 s

% 50 80 25 80 35 80

FIG. 5. Antibody-ribosome complex formation in sucrose gradients. A, dimer formation (percent) of cross-linked 30 S (0-O) and 70 S (A-A) ribosomes reacted with anti-Sl, plotted against IgG concentration. B, monomer reduction (percent) of cross- linked 30 S (a-O), 30 S(-Sl) (m-m), and 70 S (A-A) ribosomes reacted with anti-Sl. C, inhibition of monomer reduction by prein- cubation of anti-S1 with single protein Sl (Cl-Cl), of anti-Sir*. with fragment Sl-F2a (A-A), and of anti-Slrzb with fragment Sl-F2b (O-O). Dimer formation and monomer reduction was determined by planimetry (see “Experimental Procedures”).

itation is only possible if the antibodies can bind to at least three epitopes on a single ribosomal particle.

Reactivity of the fragment-specific antibodies with cross- linked 30 S subunits was similar as described above for anti- Sl; dimer formation was very low and achieved maximally 7% for anti-SIFz. and 4% for anti-S&s (Table I). As with anti-Sl, no dimer formation was seen at higher antibody concentration; however, a significant reduction of the mon- omer peak occurred (25% for anti-S1rzs and 35% for anti- S1r2b, see Table I).

When either of the Sl-specific antibodies was tested for their reactivity with cross-linked 70 S ribosomes, an unusual phenomenon was observed, in all cases both dimer formation and monomer reduction was 1.5- to 3-fold higher than with 30 S subunits (see Fig. 5, A and B, and Table 1). The possibility that this phenomenon was due to reactivity of the antibodies with epitopes on the surface of the 50 S subunit could be excluded by testing the reactivity of anti-Sl, anti- Slide, and anti-S1rzb with cross-linked 50 S ribosomes; no reactivity could be detected by sucrose gradient centrifugation (data not shown). No antibody tested previously in our labo- ratory has been more reactive with 70 S monosomes than with ribosomal subunits, and we do not know the explanation for this unusual behavior of all three Sl-specific antibodies.

Specificity Controls-As has been discussed previously (Stoffler and Stoffler-Meilicke, 1984), it is not sufficient to demonstrate the specificity of an antibody for the isolated antigen, but in addition it is necessary to demonstrate the specificity of an antibody with its antigen in situ if an antibody is to be used to localize a protein on the ribosomal surface.

In a first set of experiments all three Sl-specific antibodies were tested for their reactivity with cross-linked 30 S ribo- somes that lacked protein Sl. The absence of protein Sl from the 30 S(-Sl) particles had been demonstrated by SDS poly- acrylamide gel electrophoresis (Fig. 3, lane 4) and it had furthermore been shown that no protein other than Sl had been removed from the 30 S subunits (Fig. 3, lane 5). Upon sucrose gradient centrifugation neither of the three antibodies showed any reactivity with 30 S(-Sl) ribosomes, as illustrated for anti-S1 (Fig. 5B). These experiments demonstrated that in the intact 30 S subunit each of the three antibodies reacted exclusively with protein Sl and no other ribosomal protein.

In a second set of experiments, anti-Sl, anti-SL, and anti-S1r2b were preabsorbed with their respective antigen; reaction of the antibodies with their cognate antigen in situ was competitively inhibited by preincubation of the antibody with increasing amounts of the isolated antigen; 150 pmol of protein Sl, fragment Sl-F2a, and fragment Sl-FSb, respec- tively, completely abolished reactivity of the antibodies with

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Localization of Ribosomal Protein Sl

crosslinked 30 S subunits (Fig. 5C). This latter experiment clearly demonstrated the fragment specificity of anti-S1r2, and anti-Slr21,.

Electron Microscopy-When 30 S subunits reacted with any of the Sl-specific antibodies were prepared for electron mi- croscopy, many of the 30 S-30 S dimers had no antibody molecule(s) bound (Fig. 6A). This was expected, since the dimer peaks obtained from the sucrose gradients not only contained Sl immunocomplexes but in addition 30 S-30 S dimers generated by the cross-linking procedure. An electron micrograph of 30 S-30 S dimers prepared from the dimer peak of the 30 S control is shown in Fig. 6B for comparison. The ratio of cross-linked 30 S-30 S dimers to 30 S-IgG-30 S immunocomplexes as determined from the electron micro- graphs was approximately the same as determined from the sedimentation profiles described above. For the determination of the anti-S1 binding sites, only those 30 S-IgG and 30 S- IgG-30 S complexes were evaluated in which antibody mole- cule(s) were clearly seen to be bound to the ribosomal particle.

Localization of the NH2-terminal Domain of Sl-A general field of 30 S ribosomes reacted with anti-S1rn, is shown in Fig. 7A. Whereas approximately 15% of the 30 S-IgG-30 S dimers observed had three or more IgG molecules attached to them (see Fig. 7, e-h), 85% of the dimers had only one or two IgG molecules bound. Our interpretation of the three-dimen- sional location of the NH2-terminal domain of Sl is solely based on these latter specimens. For the three-dimensional location, the antibody binding sites were determined on the three characteristic views of the 30 S subunit which have been described elsewhere (Stbffler-Meilicke et al., 1984).

In the immunocomplexes, the majority (about 70%) of the 30 S subunits was seen in the angled asymmetric projection and antibody binding was in the region of the large lobe (Fig. 7, i-p). Sometimes, antibody binding appeared just below the tip of the large lobe (Fig. 7, m and n) or at the lower part of the head (Fig. 7, o andp). However, in all cases the Fab arms of the connecting IgG molecules were only partly visible, thus we concluded that in the angled asymmetric projection the actual antibody attachment site was away from the contour line of the large lobe, close to the cleft region of the 30 S particle (Fig. 7,b).

In the cloven asymmetric projection, which was extremely rare in the immunocomplexes (<5%), antibody binding was equally seen at the large lobe (Fig. 7, c$ q, and r). In the

FE. 6. Electron micrographs of crosslinked 30 S subunits, reacted with anti-S1 (A), and crosslinked 30 S ribosomal subunits taken from the dimer peak of the control shown in Fig. 4A (B). 30 S-IgG-30 S immunocomplexes on which bound antibody molecules can be clearly discerned are marked by arrows. Open arrowheads mark 30 S-30 S dimers that have been cross-linked to each other by the cross-linking reagent.

FIG. 7. Electron micrographs of 30 S subunits reacted with IgG antibodies specific for the NH*-terminal fragment of Sl. a, general field; b-u, selected views. The interpretative schemes rep- resent the micrograph on their immediate left.

unit. The numbers give the locations of antibody-binding sites for individual ribosomal proteins as determined in our laboratory (Stof- fler-Meilicke and Stdffler, 1987). The location of the NH2-terminal fragment (l-193) of 31 is shown shaded.

quasisymmetric view the binding site of the antibody was at the body just below the one-third/two-thirds partition (see Fig. 7, c and s-u). In most of the quasisymmetric projections the large lobe could not be clearly discerned and therefore a precise location of the antibody attachment site was not possible (Fig. 7, s-u). However, in a few quasisymmetric views the binding site of the IgG molecule was clearly on the side of the large lobe (see Fig. 7, c and u). Taking together the above data, the NH2-terminal domain of Sl has been localized at a unique site on the three-dimensional model of the 30 S ribosomal subunit, as shown in Fig. 8.

Localization of the COOH-terminal Domain of Sl-As seen with anti-Slrza, the angled asymmetric projection prevailed in the immunocomplexes obtained with anti-S1F2b, and the cloven asymmetric projection was extremely rare. The major- ity of the immunocomplexes had three or even more IgG molecules bound (see Fig. 9, a-g). Antibody binding was predominantly found in the region of the large lobe (Fig. 9, b-e), but in addition well below the large lobe (Fig. 9, b) and also on the head of the 30 S subunit (Fig. 9, c, d, f, and g). However, an interpretation of these electron micrographs was difficult due to the multiple antibodies bound. When we investigated immunocomplexes that had only one or two IgG molecules bound, we also observed that the majority of anti-

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

11342

A

,

t

4

Localization of Ribosomal Protein Sl

b c

d E

1

I m

0 P 9 *-

FIG. 9. Electron micrographs of 30 S subunits reacted with IgG antibodies specific for the COOH-terminal fragment of S 1. n. general field; b-s, selected views.

bodies were attached to the large lobe (about 55%, see Fig. 9, h-m). However, almost 40% of the antibodies bound to the head of the subunit (Fig. 9, n-s), predominantly on the side of the large lobe.

From the above electron micrographs it was not possible to determine a confined binding site for the anti-SlpLl, antibody, since epitopes of the COOH-terminal domain of protein Sl were localized at sites all over the ribosomal surface or even some distance away from it (Fig. 9, b-g). The antibody binding pattern was very similar to the one seen with anti-L7/12 (Schaber et al., 1984), and the immunoelectron microscopic data suggested that the COOH-terminal domain of Sl may be considerably elongated.

Since the complex binding pattern obtained with anti-S1F2i, did not allow us to locate the COOH-terminal domain of Sl, monovalent Fab fragments were prepared from the Sl-FBb- specific IgG and reacted with cross-linked 30 S subunits (see “Experimental Procedures”). Since monovalent Fab mole- cules bind a single epitope only, this approach should allow us to better resolve the progression of the COOH-terminal domain within the ribosomal subunit.

A typical field of cross-linked 30 S ribosomes incubated with Fab fragments prepared from anti-Slp2i, is shown in Fig. 10, a. Of the 309 immunocomplexes that have been evaluated, 47% had one (Fig. 10, c-f), 45% two (Fig. 10, g-l), 6% three (Fig. 10, m-p), and 2% four or even more Fab molecules bound (Fig. 10, q, and Fig. 11). Maximally seven Fabs bound simul- taneously to a single subunit could be discerned (Fig. 11, h).

Approximately 45% of the 30 S subunits in the immuno- complexes were seen in the angled asymmetric projection, 35% in the quasisymmetric projection, and 20% in the cloven asymmetric projection. On all three projectional forms the majority of the Fab molecules (>90%) bound on the side of the large lobe in a region extending from the head to just below the lobe, as indicated in the scheme in Fig. 10, b. Antibody binding to sites other than the ones described was seen in less than 10% of the immunocomplexes.

Immunocomplexes in which a single 30 S subunit had three or more Fabs bound simultaneously, however, illustrated that in these complexes the COOH-terminal domain of Sl was elongated (Fig. 10, m and o, and Fig. 11). Although the elongated structure of Sl cannot be visualized on the electron micrographs, we inferred from the arrangement of the Fab

k I m ” 0 P q

FIG. 10. Electron micrographs of 30 S subunits reacted with monovalent Fabs specific for the COOH-terminal fragment of Sl. a, general field; c-y, selected views. The shaded area on the scheme in b indicates the region where antibody binding has predom- inantly been observed.

b c d

h

FIG. 11. Selected electron micrographs of 30 S subunits reacted with Fabs specific for the COOH-terminal part of Sl and three-dimensional model of the 30 S subunit. a-h, electron micrographs; the interpretative schemes represent the electron mi- crographs to their immediate left. Although protein Sl cannot be directly visualized on the electron micrographs, Sl is represented by a black line in the schemes, in order to better illustrate the progression of Sl which has been deduced from the binding pattern of the Fab molecules. (i) Schematic representation of the COOH-terminal do- main of Sl (black rod) in relationship to the position of the NH,- terminal domain of Sl (black circle denoted N) on the three-dimen- sional model of the 30 S subunit. The arrow indicate that the COOH- terminal domain of Sl is flexible, at least to a certain degree.

molecules that they are aligned along the COOH-terminal part of Sl as depicted by the black line in the schemes (Fig. 11, e-h). From these electron micrographs we thus concluded that the COOH-terminal fragment of Sl extends from the ribosomal surface into the surrounding space. Considering that the width of a Fab molecule is approximately 4 nm, we estimated that the COOH-terminal domain of Sl extends at least 10 nm away from the contour line of the 30 S particle. Comparison of the electron micrographs in Fig. 10, m and p, and in Fig. 11, e and f, furthermore suggests that the COOH- terminal domain of Sl is flexible, at least to some degree, as illustrated in Fig. 11, i.

Our finding that binding of monovalent Fabs is seen in a region extending from the head to below the large lobe (see

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Localization of Ribosomal Protein Sl 11343

Fig. 10, b) does, thus, not mean that the COOH-terminal domain of Sl covers this whole area, but that the elongated part of Sl points into different directions in the various complexes. With this our interpretation in mind that protein Sl is flexible and extends from the ribosomal surface into the surrounding space, it is also possible to understand the com- plex binding pattern seen with the bivalent IgG molecules (Fig. 9). From the electron micrographs shown in Fig. 9, b-g, it would even be conceivable that protein Sl is more elongated and more flexible than shown in the scheme (Fig. 11, i).

DISCUSSION

In this study we sought to determine the locations of the NH,- and COOH-terminal domains of protein Sl on the surface of the 30 S ribosomal subunit of E. coli, using frag- ment-specific antibodies in combination with IEM. Because of the weak association of Sl with the ribosome, protein Sl had to be chemically cross-linked to the 30 S subunit in order to obtain stable 30 S-IgG-30 S immunocomplexes that were suitable for IEM. As cross-linking reagent the cleavable DSP was employed, cross-linking protein Sl to the 30 S subunit with an efficiency of almost 100%. Although the cross-linking procedure did not recognizably alter the overall structure of the ribosome as judged by electron microscopy, more subtle alterations within the ribosomal structure cannot be definitely excluded. Yet comparison of the data from a recent cross- linking study of the proteins within the 50 S ribosomal subunit (Walleczek et al., 1989) with the corresponding three-dimen- sional model derived from IEM (Stoffler and Stbffler-Meil- icke, 1986) demonstrated that the two independent sets of data were fully compatible (Walleczek et al., 1988), indicating that the cross-linking procedures do not induce major struc- tural changes within the ribosome.

Although with all Sl-specific antibodies dimer formation of cross-linked 30 S subunits was very low, a significant reduc- tion of the monomer peak was observed with increasing antibody concentrations, indicating that the ribosomes had been precipitated and that thus both the NH,- and the COOH- terminal domain of Sl had several antigenic determinants accessible for antibody binding. Because of the low dimer formation, monomer reduction was taken as measure for the specificity controls.

When we compared the reactivity of the Sl-specific anti- bodies with 30 S subunits and 70 S monosomes, a surprising observation was made, the reactivity of the antibodies was up to 3-fold higher with 70 S monosomes than with 30 S subunits, a finding which had never been observed before for any other ribosomal protein. This phenomenon was neither due to re- activity of the Sl-specific antibodies with epitopes present on the surface of the 50 S subunit nor was it due to significant differences in the Sl stoichiometry of 70 S monosomes and 30 S subunits, as judged from SDS polyacrylamide gels and immunoblotting (data not shown). Thus the most likely inter- pretation for the increased reactivity of the Sl-specific anti- bodies with 70 S monosomes is that protein Sl has different conformational states in 30 and 70 S ribosomes.

The NHz-terminal domain of protein Sl has been localized on the large lobe of the 30 S ribosomal subunit, close to the one-third/two-thirds partition, on the side which in the 70 S ribosome faces the cytoplasm (Fig. 8). Epitopes of the COOH- terminal domain have also been located in this same region, but in addition our electron micrographs provided evidence that the COOH-terminal domain of Sl is an elongated struc- ture which extends at least 10 nm away from the ribosomal surface (Fig. 11). Our data furthermore suggest that this elongated structure is flexible.

From our experiments we cannot completely exclude that the antibody molecules unfold the COOH-terminal part of Sl rather than bind to an existing elongated structure. As a matter of fact, Berestowskaya et al. (1988) came to such a conclusion when studying the structure of protein Sl within Qa replicase, and from these results the authors concluded that protein Sl also has a globular conformation within the native ribosome. However, since Berestowskaya et al. (1988) did not cross-link protein Sl to the other components of Qa replicase, they cannot differentiate whether their enzyme structure was disrupted due to the removal of protein Sl from the complex or due to unfolding of Sl by the antibody. Protein Sl contains altogether 43 lysines of which the NH*-terminal fragment contains 13 and the COOH-terminal fragment 17 (Kimura et al., 1982; Schnier and Isono, 1982). If Sl was indeed globular in situ, it is highly probable that the lysine- specific DSP which gave 100% cross-linking of Sl to the ribosome would also have induced sufficient intramolecular cross-links to prevent unfolding of Sl by the antibody.

Our finding that protein Sl is elongated and flexible in situ is consistent with studies from a number of other research groups. From neutron scattering experiments (Sillers and Moore, 1981) and fluorescence anisotropy measurements (Odom et al., 1984) it has been concluded that protein Sl is not only elongated in solution, but also when it is bound to the ribosome. Studies with proton nuclear magnetic resonance have shown that protein Sl contains a “substantial flexible component” in situ (Cowgill et al., 1984). Using Fab fragments specific for the COOH-terminal part of Sl, we could visualize the progression of the COOH-terminal domain into the sur- rounding space.

It has been shown that in solution protein Sl is 23 nm long (Laughrea and Moore, 1977; Giri and Subramanian, 1977; Yokota et al., 1977; Labischinski and Subramanian, 1979). Taking into account that the COOH-terminal domain of Sl extends at least 10 nm from the contour of the 30 S subunit, our minimal estimate of the total length of Sl in situ is approximately 15 nm (Fig. 11, i). However, from our experi- ments it cannot be excluded that protein Sl measures 23 nm even in situ. As a matter of fact, some electron micrographs (see Figs. 9, b-g, and 11, a-d, as examples) do suggest that protein Sl is more flexible and more elongated than shown in the scheme in Fig. 11, i.

A location for protein Sl has also come from neutron scattering (Sillers and Moore, 1981; Cape1 et al., 1988). In view of the finding that protein Sl is elongated and flexible in situ, the center of mass does not determine precisely the position of Sl. However, the location of Sl as determined by neutron scattering is in proximity to the location of the NH2- terminal domain of Sl as determined in this study. In contrast to neutron scattering we were able to differentiate between the locations of the NH*- and COOH-terminal domains of Sl, thus yielding a more detailed understanding of the topography of protein Sl within the ribosomal particle.

Evidence that protein Sl is located in the region of the large lobe also comes from Sl RNA binding and Sl-RNA cross-linking studies: (i) Sl was found to bind within the terminal 49 nucleotides of the 3’-end of 16 S RNA (Dahlberg and Dahlberg, 1975); the 3’-end of 16 S RNA and the dimethyl adenosines at positions 24 and 25 from the 3’-end have been localized at the large lobe by IEM (for references see Stoffler and Stoffler-Meilicke, 1986). (ii) Protein Sl, together with protein S21, has been cross-linked in situ near the 3’-end of 16 S RNA (Czernilofsky et al., 1975)). (iii) Sl has been cross- linked to nucleotides 861-889 (Golinska et al., 1981), in a region which is in the immediate neighbourhood of the site

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

11344 Localization of Ribosomal Protein Sl

where protein S18 cross-links to the 16 S RNA (nucleotides 845-851; Greuer et al., 1987); protein S18 has also been localized in the lobe region (Stoffler-Meilicke and Stoffler, 1987). All these data are in agreement with our location of the NH2-terminal region of Sl on the large lobe, close to the cleft of the 30 S subunit.

It is of interest to correlate our surface locations for the NH2- and COOH-terminal domains of Sl with studies that reveal the different functions of these two domains in uitro. The NH*-terminal domain has a high affinity for the 30 S ribosome and the COOH-terminal domain for mRNA, but not for ribosomes (see the Introduction). Against the background of these functional data, our results show that binding of Sl to the ribosome via its NHz-terminal domain takes place at the large lobe of the 30 S subunit. Our finding that the COOH- terminal domain of Sl extends into the cytoplasmic space is in agreement with the observation that this part of Sl has no affinity for the 30 S ribosome.

Finally, we discuss our findings in the light of the possible function of Sl during translation initiation. Whereas many groups have established that Sl is essential for the efficient translation of mRNA during protein biosynthesis (for a review see Subramanian, 1984), the precise mechanism of action of Sl remains to be elucidated. Subramanian (1983) proposed that the COOH-terminal domain of Sl forms a flexible lasso- like appendage. Thus his model explains how Sl (i) specifi- cally binds mRNA in the cytoplasmic space and (ii) brings the bound mRNA molecule close to the decoding site during the initiation phase of translation. Our results from IEM support such a model since they show that the COOH-termi- nal domain of Sl extends considerably from the ribosomal surface and is flexible in situ. Thus protein Sl could indeed bind and transfer mRNA from the immediate ribosomal en- vironment to the ribosomal decoding site.

Acknowledgments-We are grateful to Dr. H. G. Wittmann for his continued support and to Dr. A. R. Subramanian for providing fragments Sl-F2a and Sl-F2b for immunization. We thank R. Has- enbank and K. H. Rak for expert technical assistance.

REFERENCES

Berestowskaya, N. H., Vasiliev, V. D., Volkov, A. A., and Chetverin, A. B. (1988) FEBS. Lett. 228, 263-267

Breitenreuter, G., Lotti, M., Stoffler-Meilicke, M., and Stoffler, G. (1984) Mol. & Gen. Genet. 197,189-195

Capel, M. S., Kjeldgaard, M., Engelman, D. M., and Moore, P. B. (1988) J. Mol. Biol. 200, 65-87

Chu, Y. G., and Cantor, C.-R. (1979) Nucleic Acids Res. 6,2363-2379 Cowcill. C. A.. Nichols. B. G.. Kennv. J. W.. Butler. P.. Bradburv. E.

MT, and Tram, R. R: (1984) J. Sic% Chem. 259,15257-15263” Czernilofsky, A. P., Kurland, C. G., and Stoffler, G. (1975) FEBS

Lett. 58,281-289 Dahlberg, A. E., and Dahlberg, J. E. (1975) Proc. N&l. Acad. Sci. U.

S. A. 72,2940-2944 - Draper, D. E., and von Hippel, P. H. (1978) J. Mol. Biol. 122, 321-

338 Giri, L., and Subramanian, A. R. (1977) FEBS Lett. 81, 199-203 Golinska, B., Millon, R., Backendorf, C., Olomucki, M., Ebel, J. P.,

and Ehresmann, B. (1981) Eur. J. Biochem. 115,479-484 Greuer, B., Osswald, M., Brimacombe, R., and Stoffler, G. (1987)

Nucleic Acids Res. 15,3241-3255

Hjelm, H., Hjelm, K., and Sjoquist, J. (1972) FEBS Z.&t. 28, 73-76 Kimura, M., Foulaki, K., Subramanian, A. R., and Wittmann-Liebold,

B. (1982) Eur. J. Biochem. 123,37-53 Kitakawa, M., and Isono, K. (1982) Mol. & Gen. Genet. 185, 445-

447 Labischinski, M., and Subramanian, A. R. (1979) Eur. J. Biochem.

95,359-366 Laemmli, U. K. (1970) Nature 227,680-685 Laughrea, M., and Moore, P. B. (1977) J. Mol. Biol. 112,399-421 Leboy, P. S., Cox, E. C., and Flask, J. G. (1964) Proc. Natl. Acad. Sci.

U. S. A. 52, 1367-1374 Lomant, A. J., and Fairbanks, G. (1976) J. Mol. Biol. 104, 243-261 Maschler, R. (1973) Immunochemische Untersuchungen zur Funk-

tion der Einzeloroteine der 305 Untereinheit von Escherichia coli Ribosomen. Ph:D. thesis, Freie Universitat Berlin, Federal Repub- lic of Germany

Moore, P. B., and Laughrea, M. (1979) Nucleic Acids Res. 6, 2355- 2361

Noll, M., Hapke, B., Schreier, M. H., and Noll, H. (1973) J. Mol. Biol. 75,281-294

Odom, 0. W., Deng, H.-Y., Subramanian, A. R., and Hardesty, B. (1984) Arch. Biochem. Biophys. 230,1’78-193

Putnam, F. W., Tab, M., Lynn, L. T., Easlev, C. W., and Miaita, S. (1962) J. Bioi. Chem. 23+, 717-726 -. -

Schaber. E.. Kastner. B.. Stoffler-Meilicke. M.. and Stoffler. G. (1984) I I I I I

in Proceedings of the Eighth European Congress on Electron Mi- croscopy, Budapest, Vol. 2, pp. 1555-1556, Programme Committee of Eighth European Congress on Electron Microscopy, Budapest

Schnier, J., and Isono, K. (1982) Nucleic Acids Research 10, 1857- 1865

Sillers, I.-Y., and Moore, P. B. (1981) J. Mol. Biol. 153, 761-780 Stoffler, G., and Stoffler-Meilicke, M. (1984) Annu. Reu. Biophys.

Bioeng. 13,303-330 Stoffler, G., and Stoffler-Meilicke, M. (1986) in Structure, Function,

and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 28-46, Springer-Verlag, New York

Stoffler-Meilicke, M., and Stoffler, G. (1987) Biochimie 69, 1049- 1064

Stoffler-Meilicke, M., Epe, B., Woolley, P., Lotti, M., Littlechild, J., and Stoffler, G. (1984) Mol. & Gen. Genet. 197,8-18

Subramanian, A. R. (1983) Prog. Nucleic Acid Res. Mol. Biol. 28, 101-142

Subramanian, A. R. (1984) Trends Biochem. Sci. 9,491-494 Subramanian, A. R., and van Duin, J. (1977) Mol. & Gen. Genet. 158,

l-9 Subramanian, A. R., Rienhardt, P., Kimura, M., and Suryanarayana,

T. (1981) Eur. J. Biochem. 119,245-249 Suryanarayana, T., and Subramanian, A. R. (1979) J. Mol. Biol. 127,

41-54 Suryanarayana, T., and Subramanian, A. R. (1983) Biochemistry 22,

2715-2719 Szer, W., and Leffler, S. (1974) Proc. Natl. Acad. Sci. U. S. A. 71,

3611-3615 Tischendorf, G. W., Zeichhardt, H., and Stoffler, G. (1974) Mol. &

Gen. Genet. 134,187-209 Van Dieijen, G., van Knippenberg, P. H., and van Duin, J. (1976)

Eur. J. Biochem. 64,511-518 Walleczek, J., Martin, T., Redl, B., Stoffler-Meilicke, M., and Stoffler,

G. (1989) Biochemistry 28.4099-4105 Walleczek, J., Schiiler, G., Stoffler-Meilicke, M., Brimacombe, R.,

and Stoffler, G. (1988) EMBO J. 7,3571-3576 Wittmann-Liebold, B. (1986) in Structure, Function and Genetics of

Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 326-361, Sprin- ger-Verlag, New York

Yokota, T., Arai, K.-I., and Kaziro, Y. (1977) J. Biochem. (Tokyo) 82,1485-1489

Zubke, W., Stadler, H., Ehrlich, R., Stoffler, G., and Wittmann, H. G. (1977) Mol. & Gen. Genet. 158,129-139

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from

J Walleczek, R Albrecht-Ehrlich, G Stöffler and M Stöffler-Meilickeribosomal protein S1 on the surface of the 30 S subunit from Escherichia coli.Three-dimensional localization of the NH2- and carboxyl-terminal domain of

1990, 265:11338-11344.J. Biol. Chem. 

  http://www.jbc.org/content/265/19/11338Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/19/11338.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 28, 2019

http://ww

w.jbc.org/

Dow

nloaded from