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.     

Investigation of the 50 S ribosomal subunit by electron microscopy and image analysis

In electron micrographs of 50 S (large) subunits from Escherichia coli ribosomes, the highly preferred crown view is inferred to represent the roughly hemispherical particle lying with its flat or concave face against the carbon film. Single particle averaging allows the reproducible details of the crown view particle to be recognized. Multivariate image analysis shows the most variable morphological features of this view to be the two side protrusions, the L7/L12 stalk and the L1 ridge, both of which show apparent positional variations. The invariance of the features of the particle body implies that the movements of the side protrusions are not merely a result of perspective changes produced by major rotations of the particle body out of its quasistable, flat-lying position. A bending point localized on the L7/L12 stalk is conjectured to represent a functional "hinge" that may be related to the secondary/tertiary structure of the L7/L12 dimeric protein.     

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.     

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.     

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.     

Shape of protein L11 from the 50S ribosomal subunit of Escherichia coli.

Protein L11 from the 50S ribosomal subunit of Escherichia coli A19 was purified by a method using nondenaturing conditions. Its shape in solution was studied by hydrodynamic and low-angle x-ray scattering experiments. The results from both methods are in good agreement. In buffers similar to the ribosomal reconstitution buffer, the protein is monomeric at concentrations up to 3 mg/mL and has a molecular weight of 16 000-17 000. The protein molecule resembles a prolate ellipsoid with an axial ratio of 5-6:1 a radius of gyration of 34 A, and a maximal length of 150 A. From the low-angle x-ray diffraction data, a more refined model of the protein molecule has been constructed consisting of two ellipsoids joined by their long axes.     

Mutation affecting the thermolability of the 50S ribosomal subunit in Escherichia coli.

Genetic analysis of a mutation affecting the thermal response of the 50S ribosomal subunit to in vitro polyphenylalanine synthesis indicates that the gene, rit, is located near metB on the Escherichia coli chromosome and that the probable gene order is metB-rit-arg-rpo.     

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.     

Erythromycin inhibition of 50S ribosomal subunit formation in Escherichia coli cells

The effects of erythromycin on the formation of ribosomal subunits were examined in wild-type Escherichia coli cells and in an RNase E mutant strain. Pulse-chase labelling kinetics revealed a reduced rate of 50S subunit formation in both strains compared with 30S synthesis, which was unaffected by the antibiotic. Growth of cells in the presence of [14C]-erythromycin showed drug binding to 50S particles and to a 50S subunit precursor sedimenting at about 30S in sucrose gradients. Antibiotic binding to the precursor correlated with the decline in 50S formation in both strains. Erythromycin binding to the precursor showed the same 1:1 stoichiometry as binding to the 50S particle. Gel electrophoresis of rRNA from antibiotic-treated organisms revealed the presence of both 23S and 5S rRNAs in the 30S region of sucrose gradients. Hybridization with a 23S rRNA-specific probe confirmed the presence of this species of rRNA in the precursor. Eighteen 50S ribosomal proteins were associated with the precursor particle. A model is presented to account for erythromycin inhibition of 50S formation.     

Identification of the collar-like structure of the 30S ribosomal subunit from E. coli by dark field electron microscopy

We have used dark field electron microscopy to study a fragment of the small (30S) subunit of the E. coli ribosome. This fragment is almost the same size as the parent particle but RNA sequencing studies have shown it to lack, as a major constituent, a 150-nucleotide stretch at the 3' end of the rRNA, and two minor sections constituting 20 nucleotides from the 5' end and the 15 nucleotides of the sequence 687-701. The protein composition of the fragment was essentially unchanged. Samples of this material, and controls, were examined in the electron microscope after treatment with a buffered uranyl acetate solution for positive staining. Careful comparison revealed the following differences. The structural feature that we call the "collar" was missing in the fragment. Of the three parallel uranyl-staining bands that we have observed in micrographs of whole 30S subunits, the fragment consistently lacked the uppermost band. These observations identify the top uranyl-adsorbing band as being the 3' end of the ribosomal RNA and show that it can be equated with the collar-like structure.     

Three-dimensional reconstruction and averaging of 50 S ribosomal subunits of Escherichia coli from electron micrographs

The thermal stability and melting kinetics of the α-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil α-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 (M_r = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 (M_r = 16,000 to 17,000) and 24,000 to 26,000 (M_r = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The α-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range (τ_f) and the other in the millisecond range (τ_s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast (τ_f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.     

Physical properties of ribosomal proteins isolated under different conditions from the Escherichia coli 50 S subunit

Physical properties of ribosomal proteins obtained with or without denaturating agents were compared. CD measurements and NMR studies have shown that proteins L2, L19, L24 and L30 isolated under denaturing conditions have the same properties as those prepared avoiding denaturating agents. CD and NMR spectra of proteins L1, L6, L11, L23, L25 and L29 obtained by us under denaturating conditions practically coincide with the data for the same proteins reported under 'mild' conditions. These findings suggest that the differences of reported physical properties can be due to different procedures of protein renaturation rather than to the methods of their isolation.     

An efficient in vitro total reconstitution of the Escherichia coli 50S ribosomal subunit

A new, relatively simple technique for the total invitro reconstitution of E. coli 50S ribosomes has been developed. It is a two-step procedure like that previously reported by Nierhaus and Dohme [Proc. Natl. Acad. Sci. 71, 4713 (1974)], but it differs in a number of important aspects. Ribosomal RNA is prepared by direct phenol extraction of 70S particles to minimize nuclease fragmentation. A mixture of 50S proteins is prepared by acetic acid extraction and immediate removal of the acetic acid by thin film dialysis. The resulting protein mixture is soluble and stable. Separate RNA and protein fractions are mixed, incubated first at 44 degrees C in 7.5 mM Mg(2+), and then at 50 degrees C in 20 mM Mg(2+). The resulting 50S particles comigrate with native 50S particles in analytical gradients. They range from 50 to 100% active in five different functional assays. This is a fairly stringent test of the effectiveness of reconstitution since 50S particles derived from highly active vacant couples were used as a control.Images