The locations of ribosomal proteins BS8, BS9 and BS20 on the 30s subunit of Bacillus stearothermophilus ribosomes, and of BL3 and BL21 on the 50s subunit, were determined by immunoelectron microscopy. BL3 was found to lie half-way down the body of the 50s subunit on the interface side, below the L7L12 stalk, in agreement with the placement of the corresponding protein in Escherichia coli by neutron-scattering; BL21 was located at a similar position on the solvent side of the subunit, as predicted by cross-linking experiments with E. coli ribosomes. Similarly, BS8 was found in the upper region of the body of the 30s subunit on the solvent side, and BS9 on the top of the head of the subunit, also on the solvent side, both positions being in good agreement with neutron-scattering data and other immunoelcctron microscopy results. In contrast, BS20 was found to lie at the extreme base of the body of the 30s subunit; this placement is not compatible with the location of E. coli S20 by neutron-scattering but fits very plausibly with other biochemical data, such as sites of RNA-protein footprinting on 16s RNA, relating to the location of S20 inImmunoelectron microscopy (IEM) is a long-established method for investigating the distribution of individual ribosomal proteins on the surface of the ribosomal subunits. In the case of the 30s subunit from Eschericlzia coli, our laboratory (Stoffler-Meilicke and Stoffler, 1990) and others (Oakes et al., 1990) have been able to locate epitopes for the majority of the small subunit ribosomal proteins with the help of this technique; the results so far have shown a high level of agreement with the neutron-scattering results of Capel et al. (1988), who have mapped the positions of the mass centers of all 21 of the 30s subunit proteins. In the case of the 50s subunit, the data sets are far less complete, although epitopes for more than half of the proteins have been located by IEM (Stoffler-Meilicke and Stoffler, 1990) and the mass centers of seven proteins have been mapped by neutron-scattering (May et al., 1992). A preliminary model for the positions of 29 of the 50s subunit proteins has been proposed (Walleczek et al., 1988), by combining the IEM data with the results of in siru protcin-protein cross-linking experiments.The more recent IEM studies have turned to the investigation of ribosomal proteins from Bacillus srearothermophi-/us. The overall morphology of ribosomal subunits from this organism is very similar to that observed in E. coli (e.g. Van Heel and Stoffler-Meilicke, 1985). Furthermore, the ribosomal proteins show a high degree of sequence similarity to their counterparts in E. coli (Wittmann-Liebold, 1988) cases so far examined it has been demonstrated that the IEM locations of similar proteins on the ribosomal subunit surfaces are identical in the two organisms (Stoffler-Meilicke et al., 1984;Stoffler and Stoffler-Meilicke, 1986; Hack1 and Stoffler-Meilicke, 1988). This finding has two important consequences. First, it shows that the sequence similarity is indeed r...

A mutant of Escherichia coli has been isolated which lacked ribosomal proteins S17 and L29, as judged by two-dimensional gel electrophoresis. A battery of immunological tests was used to confirm this result. Ribosomes of this mutant were used as a control for the localization of proteins S17 and L29 on the surface of the ribosomal subunits of E. coli. Protein S17 has been localized on the 30s subunit body, 3 -5 nm away from the lower pole, while protein L29 is located at the back of the 50s particle on the opposite side to the interface.Mutants lacking any of a number of ribosomal proteins have been described [l, 21 and many prove useful in defining the role of individual components in the structure, function, assembly and regulation of the ribosome [3 -61. Before such mutants can be exploited, however, it is essential to be sure that the ribosomal protein is really missing. A first indication of the absence of a protein was given by two-dimensional gels of ribosomal proteins [l]. Subsequently, with many mutants we have used a battery of immunological tests to determine if a protein is really missing [2, 7 -91. Generally, only a single protein is lacking in these mutants. However, closer investigation of a mutant previously described as lacking protein L29 led to the conclusion that the spot of protein S17 was also missing. The initial failure to detect the lack of protein S17 was because the spot of this protein is very faint and migrated in gels to a position very near the stronger staining spots of proteins S15 and S16. We decided to employ a variety of immunological techniques on ribosomes from this mutant, AM111, to see if proteins S17 an L29 were indeed missing. Ribosomes of this mutant were then used as controls in experiments to pinpoint topographically proteins S17 and L29 on their respective subunits. MATERIALS AND METHODSIsolation of mutant AM1 11 has been described before [l]. Preparation of ribosomes, ribosomal subunits and postribosomal supernatant was as described [7]. Ribosomal proteins were extracted from ribosomes with acetic acid [lo]. Single ribosomal proteins S17 and L29, prepared according to Hindennach et al. [ll], were obtained from H. G. Wittmann.The immunization procedure and double immunodiffusion have been described previously [12, 131. Modified immunoelectrophoresis was performed as described [7]. Separation of 50s-subunit -IgG complexes by sucrose gradient centrifugation and absorption experiments were done as before [7, 81. Corresppondence to M. Stoffler-Meilicke, Abteilung Wittmann, Max-Planck-Institut fur Molekulare Genetik, IhnestraIje 63 -73, D-1000 Berlin 33Abbreviation. TP70, total ribosomal proteins from 70s ribosomes.Samples for electron microscopy taken directly from the sucrose gradients were applied to the grids using the doublelayer carbon technique [14] and the specimens were negatively contrasted with either 0.5% uranyl acetate or 2.5% uranyl formate. Photographs were taken in a Philips EM 301 at an instrumental magnification of 110 000, operated at 80 kV. RESULTS...

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