Lake (8), the head of the 30S subunit is oriented toward the central protuberance. In addition, Lake postulates a conformational

Transcription

1 Proc. Natd Acad. Sci. USA Vol. 78, No. 11, pp , November 1981 Biochemistry Arrangement, of the subunits in the ribosome of Escherichia coli: Demonstration by immunoelectron microscopy (ribosoma-topography/ribosome model/localization of ribosomal proteins, LI, and L7/L12/IgG antibodies to ribosomal proteins) BERTHOLD KASTNER, MARINA STOFFLER-MEILICKE, AND GEORG STOFFLER* Max-Planck-Institut fur Molekulare Genetik, Abteilung Wittmann, D-1000 Berlin-Dahlem, Federal Republic of Germany Communicated by Wolfgang Beermann, June 29, 1981.ABSTRACT The three-dimensional locations of Escherichia coli ribosomal proteins, Li, and L7/L12 on the surface of ribosomal subunits and 70S monomeric ribosomes were determined by electron microscopy of antibody-labeled ribosomal particles. A new approach to orient the subunits within 70S ribosomes was developed that used 30S-70S5SOS triples that were prepared by. simultaneous combination with one antibody directed against a 30S protein and another directed against a 50S protein. Electron microscope studies of triples obtained with the antibody combinations anti-/anti-li and anti-/anti-l7/l12 showed that, in 70S monomeric ribosomes, the head of-the 30S subunit is proximate to protein LI and the peptidyl transferase center but far from the rod-like appendage containing proteins L7 and L12. Electron microscopy continues to be an important approach to determining the structure ofthe ribosome. Images ofribosomes and ribosomal subunits obtainqd by negative contrasting and heavy-metal shadowing reveal a great deal of similarity in their gross shape, independent of the contrasting procedure used. Various three-dimensional ribosome models have been proposed on the basis of such electron micrographs (1-16). Although these models show considerable similarity, there are discrepancies among them that are greater than would be expected from the similarities between the electron micrographic images. The general consensus is that both ribosomal subunits have asymmetric structures, but the situation regarding the orientation of the subunits within the 70S ribosome is less clear. Electron micrographs of 70S monomeric ribosomes show a large number of different projections, revealing globular, polygonal, or slightly prolate contours and various regions of accumulated stain. In some projections, the contours of both subunits can be clearly discerned (nonoverlap views), whereas other projections show the contours of the 50S subunit superposed on the 30S subunit or vice versa (overlap views). In contrast to ribosomal subunits, 70S ribosomes lack highly distinctive structural features (7-9). All of the 70S models accept that the indentation and the lobes (or the platform) ofthe 30S subunit face the notched side of the 50S particle. The models of 70S ribosomes differ, however, with respect to the relative orientation ofthe ribosomal subunits within the monomeric ribosome (see Fig. 5). In our model (7, 16), the long axis ofthe 30S subunit is arranged horizontally, transverse to the central protuberance of the 50S subunit, with the head of the 30S subunit proximate to the wider lateral protuberance. In the model of Boublik et al (9), the 30S subunit is also arranged horizontally, but transverse in the opposite direction with the head facing the elongated side crest (i.e., the rod-like appendage). In the model of The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact Lake (8), the head of the 30S subunit is oriented toward the central protuberance. In addition, Lake postulates a conformational change in the rod-like appendage occurring during the association of subunits to 70S ribosomes (12). In this paper, we describe attempts to orient subunits within 70S ribosomes by using antibodies as labels, one directed against a 30S protein and the other against a 50S protein. For this purpose, we selected antibodies against protein and against two proteins of the 50S subunit-i.e., anti-li and anti- L7/L12-because all of these antibodies react at distinctive structural features on the ribosomal subunits. Electron micrographs were screened for 30S-70S-50S triples connected by two antibody molecules. The evaluation of such complexes enabled us to orient the subunits within the 70S ribosome. These studies were not only important for-the further clarification ofthe structure of the 70S ribosome but also necessary for the mapping of functional domains and the various ligand binding sites on the monomeric ribosome. MATERIALS AND METHODS 70S ribosomal tight couples and 50S and 30S ribosomal subunits from Escherichia coli MRE 600 (Public Health Laboratory Service, CAMR, Porton, U.K.) were isolated as described.(17). Ribosomes were heat reactivated for 30 min at 37 C (17) before reaction with the antibody. Antisera to ribosomal proteins, L1, and L7/L12 were raised and characterized as described (18-21). The IgG fraction was purified from these sera as described by Stoffler et al. (22) and also by chromatography on protein A-Sepharose. Affinity chromatography was performed using proteins, L1, and L7/ L12 conjugated to CNBr-activated Sepharose C1 4B (23). Each IgG was shown to be entirely specific for the respective antigen protein by several methods, as exemplified in the case ofprotein Li (21). Ribosomal subunits and 70S ribosomes were incubated with the specific antibody as described (5, 6). The antibody concentrations were chosen such that one-fourth to one-third ofthe ribosomal particles were dimerized. The various complexes were separated from each other and from unreacted antibody by centrifugation through 10-30% sucrose gradients in 3.5 mm Mg(OAc)2/ 100 mm NH4C1/50 mm Tris-HCl, ph 7.5/7 mm 2-mercaptoethanol for 16 hr at 18,000 rpm in an SW40 rotor. Some experiments were also performed at 20 mm Mg(OAc)2 and gave similar results. The sucrose gradient fractions were used directly to prepare the specimens for electron microscopy. Negative contrasting was performed with 0.5% uranyl acetate by the "sandwich" technique as described (6). Electron micrographs were obtained with a Philips EM 301 microscope at 80 kv and a magnification of 57,000 or 110,000. * To whom reprint requests should be addressed.

2 ... ;....g... Biochemistry: Kastner et al X g; v+ S A... '' :'-A A,,..., 0 a-li a- L 7/12 Proc. Natd Acad. Sci. USA 78 (1981) 6653 D a-l1 ME ;g:.r., a - L 7/12 FIG. 1. Electron micrographs and schematic drawings showing the binding sites of antibodies to protein on 30S subunits (Upper) and of antibodies to protein Li (Middle) and to proteins L7/L12 (Lower) on 50S subunits. Electron micrographs were selected to demonstrate the apparent antibody attachment site to the main projectional forms of ribosomal subunits and hence to explain our three-dimensional localization of these proteins (see Fig. 2 b and e.). Bar = 50 nm. RESULTS AND DISCUSSION Locations of E. coli Ribosomal Proteins, LI, and L7/ L12 on the Ribosomal Subunits. We first reinvestigated the location ofthe binding sites ofthe three antibodies on ribosomal subunits. The locations of proteins, Li, and L7/L12 on ribosomal subunits are illustrated by selected electron micrographs and interpretative drawings (Fig. 1). Protein was mapped on the top of the head of the 30S subunit on the same side as the large lobe, and protein LI showed a unique antibody binding site on the wider lateral protuberance opposite the rodlike appendage. The L7/Li2-specific antibody used bound exclusively to the rod-like appendage, a structure that is also referred to as "L7/L12 stalk" or "elongated side crest." An explanation of the nomenclature used for explaining typical structural features is given in Fig. 2. The locations of these binding sites enabled us to investigate (by double-antibody labeling experiments with 30S-70S50S triples) whether in the 70S ribosome the head of the small subunit-i.e., the anti- binding site-was in proximity to the antigenic determinants of protein LI (wider lateral protuberance) or close to the determinants ofproteins L7/LI2 (rod-like appendage). The use of these three particular antibodies had the additional advantage that proteins, LI, and L7/Li2 have been localized by both our group and Lake and collaborators (Fig. 2). The location of protein was consistent with previous studies (5, 24). An antigenic determinant ofprotein Li has been localized on the wider lateral protuberance by our group (7, 21, 25, 26) and by Lake (27). Proteins L7 and Li2 were localized by Strycharz et al (28) on a unique site, the rod-like appendage. Binding of L7/L12-specific antibody to this distinct structural feature has been found by Boublik et al (29) and by ourselves H C P Ll L 7/12 Li L7/12 B i $13 ftj. A. A.t LsV a LL b... i~ $j L-..1..i 13 c d e 7/12 FIG. 2. Locations of proteins, L1, and L7/L12. (a) Typical structural features of 30S ribosomal subunits in the asymmetric (Upper) and quasisymmetric (Lower) projection. H, head; B, body; LL, large lobe (platform); Ls, small lobe. (b and c) Stoffler (5) and Lake (24), respectively, models for three-dimensional localization of protein (6) on the 30S subunit. (d) Typical structural features of 50S ribosomal subunits in the crownshaped (Upper) and kidney-shaped (Lower) projection. A, rod-like appendage (elongated side crest); C, central protuberance (middle crest); P, wider lateral protuberance (less extended side crest); N, notch. (e and f) Stdffler (7, 11, 21, 25, 26) and Lake (27, 28), respectively, models for three-dimensional localization of protein Li (W and proteins L7/L12 (O on the 50S subunit.

3 6654 Biochemistry: Kastner et al Proc. Nad Acad. Sci. USA 78 (1981) f4u ft 'ant~~~~~~~~~~~~~~~~~~~~~~~o a - L JU",A NO A a I a - L 7/12 ARM FIG. 3. Electron micrographs and schematic drawings of 70&Ssubunit complexes (a) and 7OS-70S dimers (b) obtained with anti- (Upper), anti- Li (Middle), and anti-l7/l12 (Lower) showing the antibody binding sites on distinctive structural features of the various subunits and their rough placement on 70S ribosomes. Bar = 50 nm. (11, 26). Additional locations of proteins L7/L12 at.different positions on the 50S subunit have also been suggested (7, 29). The latter findings were, however, irrelevant to this study because the anti-l7/l12 preparations used here bound exclusively to the rod-like appendage (Figs. 1 and 2). Locations of E. coli Ribosomal Proteins, LI, and L7/ L12 on 70S Ribosomes. We had to determine whether the binding sites of these three protein-specific antibodies were accessible in 70S ribosomes. This could be tested by (i) incubating anti- with a mixture of 70S ribosomes and 30S ribosomal subunits or (ii) incubating anti-li or anti-l7/l12 with mixtures of 70S ribosomes and 50S ribosomal subunits. Electron micrographs of such 7OS 30S or 7OSSOS heterodimers (Fig. 3a) showed that all three binding sites were also accessible in 70S ribosomes. The identical specificities of the two Fab arms ofan IgG molecule guaranteed that the poorly defined antibody binding area on the 70S ribosome was indeed identical with the distinctive antibody binding site on the respective ribosomal subunit. 7OS 70S antibody complexes were also found in each of the three preparations (Fig. 3b), and we have included them to illustrate the difficulties of localizing antibody binding sites on monomeric ribosomes. The Mutual Orientation of the Ribosomal Subunits in the 70S Ribosome. The experiments described above satisfied the prerequisites required for the determination of the orientation of the subunits. 3OS 70S50S triples were formed with two different antibody combinations-anti-/anti-l1 and anti-/ anti-l7/l12-and investigated in the electron microscope. The triples with anti-/anti-li (Fig. 4a-c) demonstrated that these two binding sites were very close in the 70S ribosome. In contrast, protein and proteins L7/,12 were far apart in the 70S ribosome (Fig. 4d-f). The interpretative drawings illustrate the conclusions that can be drawn with regard to the mutual orientation of the two subunits in the monomeric ribosome: The head of the small subunit was proximal to the Li protuberance but far away from the L7/L12 appendage (Fig. 4candf). This subunit orientation is incompatible with the subunit arrangement proposed by Boublik et al (9) (Fig. 5c). In their model, proteins and L7/Li2 (the 30S head and the extended 50S subunit side crest) are close (Fig. Sc), which contradicts the double-antibody labeling results shown in Fig. 4. Although the 70S model of Lake (Fig. Sb) and that proposed by ourselves (Fig. Sa) are different, both models are consistent with the two experiments shown in Fig. 4. In our model, and Li are closer to each other and and L7/L12 are farther apart than in the model of Lake (see Fig. 5 a and b). However, it cannot be excluded that antibody sites that appear to be close on two-dimensional electron microscopic images are in fact not as close to each other in three dimensions. Therefore, the double-antibody labeling procedure is more reliable for determinning which proteins are far apart than for determining which proteins are close together. The distance between the anti- and the anti-l7/l12 binding sites in 70S ribosomes of33 triples was measured and found to be 20 ± 4 nm. This distance is in better agreement with the 70S model proposed by our group (7, 16, 26) than with that of Lake (8, 12). These data should, however, not be overinterpreted, because antibody specific to proteins L7/L12 bound on multiple sites on the rod-like appendage (Fig. ic), which is -8 nm long. A histogram made from the measured -L7/L12 distances in fact gave two different maxima (unpublished results). Additional binding to 3OS 70S-50S triples ofa monovalent Fab fragment specific for a third protein should facilitate these studies, because ribosome-bound Fab can be clearly visualized (see figure 9 in ref. 11). These experiments should eventually enable us to discriminate between our model and that of Lake. The kind of experiments described here offer two possibilities. First, they should allow the exact determination of the mutual orientation of the subunits in the ribosome without reference to the observed shapes of 70S particles. Second, the question of the detailed shape of the 70S particle (7-9, 16, 26) should be resolvable by further evaluation of 30S 70S, 5OS 70S, and 7OS 70S antibody complexes (see Fig. 3). Knowledge of the complete 70S structure, including the mutual orientation of the subunits in the ribosome, is also a prerequisite for studies of functional domains. Functional domains, such as the 30S ribosomal decoding site and the peptidyl transferase center, the binding sites of initiation factor IF-3, of trna, and of antibiotics, have all been mapped on ribosomal subunits (11, 16, 30-33). It is obviously important to be able

4 Biochemistry: Kastner et al Proc. NatL Acad. Sci. USA 78 (1981) 6655 gk-:,~~~~~~~~~~~~~~~~~~~~~~ N-03.M g.~~~ ~~~~~ ~~~ ~~~ ~~~ ~~~ ~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ N af.!.- *jt. br a. -a - 9 *..~.... alt FIG. 4. General field (a and d) and selected (b and e) electron micrographs of 30S&70S50S triples obtained with anti-/anti-l1 (a and b) and anti-/anti-l7/l12 (d and e). (c and f) Schematic drawings interpreting the results of the two experiments with regard to the arrangement of ribosomal particles in the resulting triples. The triples shown in b were selected from a total of 30, and those in e were selected from a total of 33. Bar = 50 nm L7/12 L7/12 L I L1 L 7/ ZL1 a b C FIG. 5. Three-dimensional models of E. coli 70S ribosomes showing the locations of proteins, L1, and L7/L12 as determined by immunoelectron microscopy. (a) Stoffler. (b) Lake. (c) Boublik.

5 6656 Biochemistry: Kastner et al to transfer these data, which have been determined experimentally on subunits, to the 70S monomeric ribosome. Other interesting sites-e.g., the binding areas ofthe elongation factor EF-Tu and the termination factor RF-2 and the site of the emerging polypeptide chain-can only be mapped on 70S ribosomes and not on subunits. The studies presented here will facilitate the more exact placement of these functional domains on 70S ribosomes. We thank Drs. R. Brimacombe, J. Littlechild, and E. R. Dabbs for frequent and valuable discussions during these experiments and the writing of this manuscript. We are grateful to Prof. H. G. Wittmann for his constant interest and support. The excellent technical expertise and assistance of Karl-Heinz Rak is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Sfb 9). 1. Lubin, M. (1968) Proc. NatL Acad. Sci. USA 61, Wabl, M. R., Barends, P. J. & Nanninga, N. (1973) Cytobiology 7, Wabl, M. R., Doberer, H. G., Hoglund, S. & Ljung, L. (1973) Cytobiology 7, Vasiliev, V. D. (1974) Acta BioL Med. Germ. 33, Tischendorf, G. W., Zeichhhardt, H. & Stoffler, G. (1974) Mol, Gen. Genet. 134, Tischendorf, G. W., Zeichhardt, H. & Stoffler, G. (1974) MoL Gen. Genet. 134, Tischendorf, G. W., Zeichhardt, H. & Stoffler, G. (1975) Proc. NatL Acad. Sci. USA 72, Lake, J. A. (1976) J. Mol BioL 105, Boublik, M., Hellmann, W. & Kleinschmidt, A. K. (1977) Cytobiology 14, Miyazawa, Y., Hosoi, J., Imamura, T. & Mochizuki, A. (1978)J. Electron Microsc. 27, Stoffler, G., Bald, R., Kastner, B., Lfihrmann, R., Stoffler-Meilicke, M., Tischendorf, G. & Tesche, B. (1979) in Ribosomes- Structure, Function and Genetics, eds. Chambliss, G., Craven, G. R., Davies, K., Davis, J., Kahan, L. & Nomura, M. (Univ. Park Press, Baltimore, MD), pp Lake, J. A. (1979) in Ribosomes-Structure, Function and Genetics, eds. Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, M. (Univ. Park Press, Baltimore, MD), pp Proc. Nad Acad. Sci. USA 78 (1981) 13. Leonard, K. R. & Lake, J. A. (1979)J. Mol Biol. 129, Spiess, E. (1979) Eur. J. Cell. Biol 19, Kom, A. P. (1980) Ultramicroscopy 5, Stoffler, C. & St6ffler-Meilicke, M. (1981) in International Cell Biology , ed. Schweiger, H. G. (Springer, New York), pp Noll, M., Hapke, B., Schreier, M. H. & Noll, H. (1973)1. Mol Biol 75, St6ffler, G. & Wittmann, H. G. (1971) Proc. Natl Acad. Sci. USA 68, St6ffler, G. & Wittmann, H. G. (1971) J. Mol Biol 62, Zubke, W., Stadler, H., Ehrlich, R., Stbffler, G., Wittmann, H. G. & Apirion, D. (1977) MoL Gen. Genet. 158, Dabbs, E. R., Ehrlich, R., Hasenbank, R., Schroeter, B.-H., Stbffler-Meilicke, M. & St6ffler, G. (1981) 1. Mol Biol 149, Stoffler, G., Hasenbank, R., Lutgehaus, M., Maschler, R., Morrison, C. A., Zeichhardt, H. & Garrett, R. A. (1973) MoL Gen. Genet. 127, Tischendorf, G. W. & Stoffler, G. (1975) Mol Gen. Genet. 142, Lake, J. A. & Kahan, L. (1975) J. Mol Biol 99, Wabl, M. R. (1974) J. Mol. Biol 84, Kastner, B., Stoffler-Meilicke, M. & Stoffler, G. (1980) in Electron Microscopy 1980-Biology, eds. Brederoo, P. & de Priester, W. (Electron Microsc. Found., Leiden), Vol. 2, pp Lake, J. A. (1978) in Gene Expression-Protein Synthesis and Control, RNA Synthesis and Control, Chromatin Structure and Function,, eds. Clark, B. F. C., Klenow, H. & Zeuthen, J. (Pergamon, New York), Vol. 43, pp Strycharz, W. A., Nomura, M. & Lake, J. A. (1978)J. Mol Biol 126, Boublik, M., Hellmann, W. & Roth, H. E. (1976) J. Mol Biol 107, Keren-Zur, M., Boublik, M. & Ofengand, J. (1979) Proc. Natl Acad. Sci. USA 76, McKuskie Olson, H., Grant, P. G., Glitz, D. G. & Cooperman, B. S. (1980) Proc. Natl Acad. Sci. USA 77, Ofengand, J. (1979) in Ribosomes-Structure, Function and Genetics, eds. Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L. & Nomura, M. (Univ. Park Press, Baltimore, MD), pp Lfihrmann, R., Bald, R., Stoffler-Meilicke, M. & St6ffler, G. (1981) Proc. Natl Acad. Sci. USA 78, in press.