Miloslav Boublik

Multivariate statistical analysis of ribosome electron micrographs: L and R lateral views of the 40 S subunit from HeLa cells

Abstract The morphology of the small (40 S) subunit of the eukaryotic ribosome from HeLa cells has been examined by single-particle averaging and multivariate image analysis applied to electron micrographs of negatively stained specimens. The use of multivariate image analysis allows different, independent components of the structural variation within the particles to be identified and separately studied. The largest component of variance for both lateral views (termed L and R) was the variation in the peripheral stain intensity. The second largest component of interparticle variation is due to changes in the particle appearance most likely associated with a change of orientation on the specimen film. Averages formed from particles falling within a small range of peripheral stain intensity allowed the changes in the projected structure to be studied as a function of local stain level. Visual observations of stain variation could be confirmed quantitatively. Significant differences were found between averages of particles in the L view and those in the R view. Multivariate image analysis of a mixed population of L and mirrored R particles showed that the differences consistently affect all particles. However, the R view increasingly resembles the L view as the overall level of stain is increased, in agreement with a model of partial stain immersion.

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

Abstract S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, E. coli rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).

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.

Localization of ribosomal proteins L6L12 in the 50 S subunit of Escherichia coli ribosomes by electron microscopy

The localization of ribosomal proteins L7L12 on the large (50 S) subunit of Escherichia coli ribosomes has been determined by electron microscopy. The results from two independent methods, labeling with antibody specific against L7L12 and depletion of the 50 S subunits of the proteins L7L12, are in agreement and show that these proteins are located in the crown-shaped region of the 50 S ribosomal subunit. The established asymmetry of the crown facilitates the topographical localization of ribosomal constituents. Proteins L7L12 are involved in several functions related to aminoacyl and peptidyl sites on the ribosome; the depletion and reconstitution experiments provide, therefore, a direct possibility of correlating the changes in the fine structure of ribosomes with their biological function.

Three-dimensional structure of 50 S Escherichia coli ribosomal subunits depleted of proteins L7/L12.

A structural study of Escherichia coli 50 S ribosomal subunits depleted selectively of proteins L7/L12 and visualized by low-dose electron microscopy has been carried out by multivariate statistical analysis, classification schemes and the new reconstruction technique from single-exposure, random-conical tilt series. This approach has allowed us to solve the three-dimensional structure of the depleted 50 S subunits at a resolution of 3 nm-1. In addition, two distinct morphological populations of subunits (cores) have been identified in the electron micrographs analyzed and have been separately studied in three dimensions. Depleted subunits in the two morphological states present as main features common to these two structures but different from those of the non-depleted subunit (1) the absence of the stalk, (2) a rearrangement of the stalk-base that changes the overall structure of this region. This morphological change is quite noticeable and important, since this region is mapped as a part of the GTPase center. The two conformations differ mainly in the orientation of the area between the L1 region and the head (the probable localization of the peptidyl transferase center) and in the accessibility of the region located below the head. A possible relationship of these structural changes to the functional dynamics of the ribosome is suggested.

Conformation of ribosomes from the vegetative amoebae and spores of Dictyostelium discoideum

SummaryRibosomes from two different cell types of D. discoideum — the undifferentiated amoebae and the differentiated spores — display considerable differences in protein composition (Ramagopal and Ennis 1979 and manuscript in preparation). These differences do not affect the three-dimensional structure of monosomes and large (60S) and small (40S) subunits from the two cell types to an extent detectable by sedimentation analysis or electron microscopy. High resolution electron microscopic images of ribosomal particles from the amoebae and the spores are similar and, in general, comparable to that of 80S, 60S and 40S ribosomal particles from other eukaryotic sources. However, distinct differences in the conformation and stability of the two types of ribosomes are detectable by circular dichroic spectroscopy. The degrce of the ordered secondary structure of rRNAs is similar in the 80S monosomes from the amoebae and the spores, but higher in the amoeba subunits. The results of thermal melting experiments show that the small subunit from the spores is more stable than that from the amoebae. The established differences in the conformation of rRNAs, most probably due to the interactions with cell-specific ribosomal proteins can be responsible for the differences in stability of ribosomes from the two cell types of slime mold.

Acetobacter bacteriophage A-1

A bacteriophage ofAcetobacter suboxydans was isolated and found to correspond to type A phage according to Bradleys classification. The phage contains double stranded DNA. The length of the latency period and burst size could not be precisely determined because of apparent non-synchronous release of phage from single infective cycles. The host range was determined using 24 strains ofAcetobacter andGluconobacter species. Evidence for a probable occurence of host determined restriction and modification was obtained withAcetobacter suboxydans strain ATCC 621. The phage is designated A-1 and it is the first one to be reported forAcetobacter.

Structure of Ribosomes and Their Components by Advanced Techniques of Electron Microscopy and Computer Image Analysis

High-resolution electron microscopy plays a leading role in the structural analysis of biological macromolecules and is the most direct method for obtaining detailed information on the morphology, topography of the components, and functional sites of ribosomes. Electron microscopy (EM) has been important also for the interpretation of data obtained by a variety of physico-chemical techniques (for references see Chambliss et al., 1980; Liljas, 1982; Wittmann, 1983) on ribosomal protein locations, relative protein-protein distances, and protein-RNA binding sites, data which are more valuable when mapped within a well-defined structural framework provided, at present, by EM.

[3] Structural analysis of ribosomes by scanning transmission electron microscopy

Publisher Summary This chapter describes that dedicated scanning transmission electron microscopy (STEM) is uniquely suited to high-resolution structural studies on ribosomes and other biological macromolecules. High efficiency in collection of scattered electrons in the STEM dark-field mode makes it possible to visualize unstained freeze-dried ribosomes and their components without the main resolution-limited artifacts of staining and distortion by air-drying and radiation inherent in the conventional transmission electron microscopy (TEM). The linearity of the relationship between scattering cross-section and molecular weight can be utilized for the determination of the molecular mass of ribosomes and their constituents, mass distribution within the particles, and calculation of the apparent radius of gyration. Protein-free deposition of unstained freeze-dried rRNA molecules improved significantly the visualization of their conformation and made it possible to initiate high-resolution studies of RNA-protein interactions and the process of ribosome assembly. Supplementation of STEM with an electron energy loss spectrometer and application of computer image averaging and multivariate statistical analysis of electron micrographs provide additional highly specific and quantitative information on three-dimensional structure, mass, and element distribution in the ribosome for topographical and phylogenetic studies.

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to a site bound transfer RNA

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.