Ribosomal proteins S3, S6, S8 and S10 of Escherichia coli localized on the external surface of the small subunit by immune electron microscopy

The three-dimensional locations of Escherichia coli ribosomal proteins S3, 86, S8 and S10 on the surface of the small subunit were determined by immune electron microscopy.All four proteins are located on the “external surface” of the small subunit; i.e. on the side of the subunit in contact with the cytosol in the 70 S ribosome. Proteins S3, S6, S8 and S10 map at single sites, although the S3 site is extended approximately 40Å along the long axis of the subunit. S8 is located near the base of the cleft separating the platform from the upper one-third or head; protein S10 is located in the head, near the site previously mapped for S14; S3 extends from the level of the constriction to near the top of the head in the vicinity of S10; and S6 is located on the platform of the small subunit near the site previously mapped for S11.The locations of these proteins correlate well with other information on their spatial relationships obtained from assembly interactions, neutron diffraction, crosslinking and protein associations.     

Positions of proteins S14, S18 and S20 in the 30 S ribosomal subunit of Escherichia coli

A map of the 30 S ribosomal subunit is presented giving the positions of 15 of its 21 proteins. The components located in the map are S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S14, S15, S18 and S20.     

The topography of ribosomal proteins on the surface of the 30S subunit of Escherichia coli

Eight ribosomal proteins, S6, S10, S11, S15, S16, S18, S19 and S21 have been localized on the surface of the 30S subunit from Escherichia coli by immuno electron microscopy. The specificity of the antibody binding sites was demonstrated by stringent absorption experiments. In addition we have reinvestigated and refined the locations of proteins S5, S13 and S14 on the ribosomal surface which had previously been localized in our laboratory (Tischendorf et al., Mol. Gen. Genet. 134, 209-223, 1974). Thus altogether 16 out of the 21 ribosomal proteins of the small subunit from E. coli have been mapped in our laboratory.     

Structure and structural variations of the Escherichia coli 30 S ribosomal subunit as revealed by three-dimensional cryo-electron microscopy

A three-dimensional reconstruction of the 30 S subunit of the Escherichia coli ribosome was obtained at 23 A resolution. Because of the improved resolution, many more structural details are seen as compared to those obtained in earlier studies. Thus, the new structure is more suitable for comparison with the 30 S subunit part of the 70 S ribosome, whose structure is already known at a better resolution. In addition, we observe relative and, to some extent, independent movements of three main structural domains of the 30 S subunit, namely head, platform and the main body, which lead to partial blurring of the reconstructed volume. An attempt to subdivide the data set into conformationally defined subsets reveals the existence of conformers in which these domains have different orientations with respect to one another. This result suggests the existence of dynamic properties of the 30 S subunit that might be required for facilitating its interactions with mRNA, tRNA and other ligands during protein biosynthesis.     

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.     

Structural studies on the 30 S ribosomal subunit from Escherichia coli

Small-angle X-ray scattering curves computed for various 30 S subunit structures have been compared with the experimental scattering curve. The curve from the 30 S subunit is best approximated by that calculated for a 1:3.6:3.6 ellipsoidal structure. The rather prolate ellipsoidal model suggested by recent electron microscope studies gives a scattering curve considerably different from the 30 S curve, suggesting that the electron microscope model is not that found in solution. Analysis of the more extended portions of the experimental scattering curve suggests some internal structure. A model is proposed that contains RNA and protein in positions such that the calculated scattering curve shows more extensive, yet similar internal structure. Resultant constraints on the structure of the 30 S subunit in solution are given.     

Direct localization of the tRNA--anticodon interaction site on the Escherichia coli 30 S ribosomal subunit by electron microscopy and computerized image averaging

Previous immunoelectron microscopy studies have shown that the anticodon of valyl-tRNA, photocrosslinked to the ribosomal P site at the C1400 residue of the 16 S RNA, is located in the vicinity of the cleft of the small ribosomal subunit of Escherichia coli. In this study we used single-particle image-averaging techniques to demonstrate that the 30 S-bound tRNA molecule can be localized directly, without the need for specific antibody markers. In agreement with the immunoelectron microscopy results, we find that the tRNA molecule appears to be located deep in the cleft of the 30 S subunit. We believe that the use of computer image averaging to localize ligands bound to ribosomes and other macromolecular complexes will become widespread because of the superior sensitivity, precision and objectivity of this technique compared with conventional immunoelectron microscopy.     

Structure of the 50S ribosomal subunit from Escherichia coli. Investigation of the intact subunit and core particles by electron microscopy and analogue image processing.

Structures of 50S ribosomal subunits, CsCl and ethidium bromide core particles from these subunits have been investigated by electron microscopy and image processing by FAIRS. This method revealed structural details which are obscured in individual images, and enabled to distinguish six crown forms, different in their side protuberances, and two kidney forms. Crown forms were imaged as symmetrical or asymmetrical forms. The latter type was far more frequent in untreated populations than the first. The depletion of proteins by both agents caused stepwise degradation of the side protuberances in the crown forms thereby transforming asymmetrical to symmetrical forms. It is concluded from these findings that asymmetrical and symmetrical forms in untreated populations represent also structurally different particles. From the higher complexity in terms of component composition and structure it is concluded that the asymmetrical crown forms are more likely to represent the native structure of isolated 50S subunits than the symmetrical forms. Existing models for this subunit are discussed in terms of this finding.     

Differential localization of two epitopes of Escherichia coli ribosomal protein L2 on the large ribosomal subunit by immune electron microscopy using monoclonal antibodies.

Two monoclonal antibodies (mAb), directed toward different epitopes of Escherichia coli ribosomal protein L2, have been used as probes in immune electron microscopy. mAb 5-186 recognizes an epitope within residues 5-186 of protein L2; it is seen to bind to 50 S subunits at or near the peptidyl transferase center, beside the subunit head on the L1 shoulder. mAb 187-272 recognizes an epitope within residues 187-272. This antibody binds to the face of the 50 S subunit, below the head and slightly toward the side with the stalk; this site is near the translocation domain. Both antibodies can bind simultaneously to single subunits. This indicates that protein L2 is elongated, reaching from the peptidyl transferase center to below the subunit head and approaching the translocational domain. The different locations of the two epitopes are consistent with previous biochemical results with the two antibodies (Nag, B., Tewari, D. S., Etchison, J. R., Sommer, A., and Traut, R. R. (1986) J. Biol. Chem. 261, 13892-13897).     

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.     

Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit

Complexes of 30 S subunits and [¹⁴C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.     

Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.     

Structure of LiCl core particles of 50 S ribosomal subunits from Escherichia coli by electron microscopy.

The structure of 50 S E. coli ribosomal subunits was studied by electron microscopy as these particles were gradually depleted of proteins by incubation with 0.5 to 6.0 m LiCl. Changes observed in the structure of the depleted subunits were correlated with the location of the deleted ribosomal proteins on the control 50 S particle. These changes were particularly striking in the "crown" region, the site of a considerable number of the proteins necessary for the biological activity of the 50 S subunit. Protein L 16, the first to be removed by the LiCl treatment, was found to be essential for the structural integrity of the large subunit through interactions with ribosomal proteins residing in the left-hand side crest and the interface. Based on electron microscopic evidence, a scheme was proposed for the structural changes accompanying the stepwise unfolding of the 50 S E. coli subunit by LiCl.     

Structure of the Escherichia coli 50 S ribosomal subunit

Freeze-dried and shadowed Escherichia coli 50 S ribosomal subunits have been examined by electron microscopy and a model of the subunit has been constructed. High resolution shadow casting has enabled us to determine independently the absolute hand of the subunit and to reveal some new structural features.