A battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.

No abstract

Antibodies were raised against eukaryotic (rat liver) and prokaryotic (E. coli) ribosomal particles and ribosomal proteins. The antisera were characterized and used to determine the identity of the eukaryotic proteins homologous to E. coli L7 and L12. The large subunit of rat liver ribosomes contains two acidic proteins, L40 and L41; they migrate during two-dimensional polyacrylamide gel electrophoresis in a way that mimics the behavior of L7 and L12. Rat liver L40 and L41 were found to be immunologically related to E. coli L7/L12, hence the proteins are likely to share some structural homology.The large subunits of Escherichia coli and other prokaryotic ribosomes contain a pair of acidic proteins which have been designated L7 and L12 from their positions on two-dimensional electropherograms (1). The proteins have been characterized and their primary sequences determined (2-7); L7 differs from L12 only in that its N-terminal serine is acetylated. There is information also on the function of the proteins, which seem essential for the binding of factors during initiation of protein synthesis and during the elongation and termination reactions (8)(9)(10)(11)(12)(13)(14).There are also two acidic proteins in rat liver 60S ribosomal subunits and they have electrophoretic properties similar to E. coli L7 and L12; the eukaryotic proteins have been designated L40 and L41 (15). We decided to attempt to determine if L7 and L12 had been conserved during evolution from prokaryotes to eukaryotes, and especially if E. coli L7 and L12 were homologous with rat liver L40 and L41. For this purpose antibodies raised against prokaryotic (E. coli) and eukaryotic (rat liver) ribosomal proteins were used. MATERIALS AND METHODSSeparation ofRibosomes and Ribosomal Particles. The following have been described before: the preparation of liver ribosomes (16) and ribosomal subunits (16, 17) from male SpragueDawley rats; the preparation of ribosomes (18) and ribosomal subunits (18, 19) from E. coli strain K12-A19 (20).Abbreviations: TP, total proteins-used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixture of all the proteins obtained from 80S ribosomes; A, antiserum or antibodies-A-TP40 is an antiserum (or antibody preparation) raised against a mixture of the proteins obtained from 40S ribosomal subunits; L7/L12, the structures of L7 and L12 are nearly identical (the N-terminal serine of L7 is acetylated), the notation, therefore, refers to a protein which is in either form; L40/ L41 is used in the same way to refer to two proteins of the large subunit of rat liver ribosomes although their primary sequence is not known.Separation of Ribosomal Protein. Proteins were extracted from ribosomes and ribosomal subunits with 67% acetic acid (16,21,22) or with 4 M urea-2 M LiCl (23). Individual E. coli ribosomal proteins were isolated and purified by carboxymethyl-cellulose chromatography and filtration through Sephadex G-100 (18, 24). Rat liver ribosomal proteins L40 and L41 were prepared by treating 2000 A2...

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...

No abstract