Immunoelectron microscopic localization of ribosomal proteins BS8, BS9, BS20, BL3 and BL21 on the surface of 30S and 50S subunits from Bacillus stearothermophilus.

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 L7/L12 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 immunoelectron 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 in E. coli.

Three-dimensional localization of the NH2- and carboxyl-terminal domain of ribosomal protein S1 on the surface of the 30 S subunit from Escherichia coli.

Antibodies were raised against Escherichia coli ribosomal protein S1 and its NH2- and COOH-terminal fragments, and their specificity was demonstrated by a variety of immunological techniques. These antibodies were then used to investigate the location of protein S1 and its NH2- and COOH-terminal domains on the surface of the 30 S ribosomal subunit by immunoelectron microscopy. In order to prevent dissociation of the protein during the experiments, S1 was cross-linked to 30 S subunits with dithiobis(succinimidyl-propionate); cross-linking yield was 100%. Epitopes of the NH2-terminal domain of S1 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-terminal part of S1 provide evidence that the COOH-terminal domain forms an elongated structure extending at least 10 nm from the large lobe of the small subunit into the cytoplasmic space.

A mutant from Escherichia coli which lacks ribosomal proteins S17 and L29 used to localize these two proteins on the ribosomal surface.

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.