Ribosome Tetramers Research Articles

In vitro crystallization of ribosomes from chick embryos.

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.

Views of Ribosome Tetramers and the Localization of Ribosomal Subunits

Carey suggested that chick-embryo ribosome tetramers attach to the substrate film preferentially in one of the two possible orientations, making them particularly appropriate for use in the three-dimensional reconstruction of ribosome structure. We have stored and analyzed tetramer images digitally as described earlier. To test for a fixed orientation, many tetramer Images were compared to a reference image that contained an arbitrarily labelled "right-handed" view. Images that correlated well with the reference were averaged, giving a right-handed result (fig. 1). To search for a left-handed view, the reference image was "turned over” in the computer to artificially create a left-handed image. Repeating the procedure with the new reference gave a result that was mostly symmetric (fig. 2), not left-handed.

Ribosome Structure Investigated by Digital Accumulation and Processing of Ribosome Tetramer Images

Electron microscopic observation of ribosomes, especially combined with 3-D reconstruction methods, may reveal structural relationships among components of the ribosome. In such studies it is necessary to average over a large number of specimen particles, to enhance the predominant structure while suppressing local irregularities of specimen preservation and staining, and to reduce the statistical insufficiencies of individual low-dose electron micrographs. One very successful method makes use of an average over the particles in a two-dimensional crystalline layer, but if the particles are not well organized, the resolution is reduced. Organization of crystalline arrays can be enhanced by computer methods, but if computational alignment is used it seems most profitable to avoid the restriction to crystalline spec- iment completely and work with isolated particles. A fixed orientation is still helpful; so in studying ribosomes, which lack their own symmetry, we have chosen to observe ribosome tetramers. The tetramers, units of the chick embryo ribosome crystal, present a fixed view in the electron micrograph.

Direct Visualization of Uranyl Stain Crystallization on a Biological Specimen During Electron Irradiation

Electron diffraction patterns from thin films of uranyl acetate (Uac) stain show that with increasing electron dose there is a narrowing of the initially broad and diffuse ring of intensity due to the stain, indicating that the stain becomes microcrystalline. The sharp intensity rings observed at higher dose have been identified as UO2 crystals. Here we report on direct visualization of the formation of 3 Å lattice fringes in the Uac stain during irradiation. The stain was used to produce negatively and positively contrasted tetramers of 80S ribosomes.The three images of negatively stained ribosome tetramers in Fig. la are part of a dose series taken at 200,000x. The focus is corresponding to minimum phase contrast. The first image was exposed to a dose of 400 e/Å2, while the second and third images were exposed to 3 and 100 times more dose, respectively. Locally high contrast regions smaller than 10 Å can be seen in any of the micrographs, but their appearance deteriorates in the subsequent micrograph of the series, indicating considerable atomic mobility.

X-ray study of the paracrystalline nature of crystallized 70Sribosome lamellae

If a mixed organic solvent is added to a ribosome solution and stored at 277 K in a crystallization chamber, thin lamellae of a few μm diameter can be obtained. Under the electron microscope, tetragonal face-centred macrocells of b = 40 and c = 55 nm are visible. These findings are substantiated by X-ray small- and wide-angle diffraction which leads to macrocells of b = 42 and c = 52 nm and establishes the third dimension of the macrocell with a = 42 nm. Mainly 0kl reflections appear. From their line widths, it follows that each lamella consists of at least 15 × 15 × 15 macrocells with small paracrystalline distortions gs - 1.5%, whilst their 6 × 8 × 12 subcells with a0 = 7.1, b0 = 5.3 and c0 = 4.3 nm have large fibrillary distortions in the bc plane, similar to the aggregation of microparacrystals in stretched and annealed polymers. With a new method, the folding root of the Q function projected on the b axis can be analysed. Two compounds are placed in each macrocell, each consisting of about 136 cylinders of about 2.0 nm diameter and 7 nm length. They are aligned to fibrils of different lengths orthogonal to the bc plane and occupy about 47% of the macrocell. The residual volume is filled up with an 'amorphous phase' as in semicrystalline polymers. A comprehensive study of the existing literature leads to the conclusion that the 'crystalline phase' consists of two 70S ribosome tetramers and that the cylinders are obviously parts of two-stranded helices, whilst most of the RNA proteins are in the 'amorphous phase'. The biological significance of the ribosome crystals and the paracrystalline sublattice is described in detail in the Discussion.

Notizen: Ribosome Crystals in the Oocyte of Gerris najas (Heteroptera)

Oocytes of the pond skater, Gerris najas, display ribosome tetramers that are arranged in the form of sheets in the vicinity of the nucleus. This is the first finding of ribosome crystals in an insect and suggests that ribosome crystallization may be a common phenomenon of cells that are inactive in protein synthesis.

The structure of ribosomes as indicated by studies of tetramers from hypothermic chick embryos.

Ribosome tetramers induced in chick embryos by exposure to cold, and tetramers of large subunits derived from them, have been studied by electron microscopy and sucrose-density-gradient analysis. Individual ribosomes of the normal tetramer are elongated bean-shaped structures, 220-280A by 195A (1A=10(-1)nm) with a cleft in the outer edge which divides the two-dimensional image into two unequal ends. Most of the tetramers appear to attach to the surface of the electron-microscope grid by one preferred face. The subunits of the large-subunit tetramers have a round outline and no cleft. About 25% of the subunits of these tetramers have a line running radially across the particle. The dissociation of tetramers into large-subunit tetramers and small subunits has been shown to be reversible. Mixtures of these particles from sucrose-density-gradient fractions were reassociated to give a tetramer with the same sedimentation coefficient as the original tetramer and with the same structure as viewed in the electron microscope. The results indicate that the cleft is a property of the complete ribosome, and that it marks the position of the small subunit. The reversibility of the dissociation also strengthens the view that no change in the large subunit occurs during dissociation or reassociation, i.e. that the sites of interaction between ribosomes in both types of tetramer are the same. The conclusions affect the interpretation of electron-micrograph images and an anomaly in the relationship between the two types of tetramer is discussed.

Ribosome crystallization in chicken embryos. I. Isolation, characterization, and in vitro activity of ribosome tetramers.

Isolated tetrameric particles (166S) derived from the crystalline lattices known to appear in hypothermic chicken embryos consist of mature 80S ribosomes which contain all species of ribosomal RNA and a complete set of ribosomal proteins. Ribosome tetramers are not a special type of polysomes since in solutions of high ionic strengths (500 mM KCl and 50 nM triethanolamine-HCl buffer) containing 5 mM MgCl(2) they dissociate into 40S and 60S ribosomal subunits, without the need of puromycin, and at a concentration of Mg(++) higher than 3 mM they are not disassembled by mild RNase treatment. Tetramers spontaneously disassemble into 80S monomers when the Mg(++) concentration is lowered to 1 mM at relatively low ionic strength. Tetramers failed to couple in vitro puromycin-(3)H into an acid-insoluble product, indicating the lack of nascent polypeptide chains. Although tetramers have no endogenous messenger RNA activity, they can be programmed in vitro with polyuridylic acid (poly U) to synthesize polyphenylalanine. All ribosomes within a tetramer can accept poly U, without the need of disassembly of the tetramers into monomers or subunits.

The location of the ribosomal small subunit in the structure of hypothermic ribosome tetramers.

The location of the ribosomal small subunit in the structure of hypothermic ribosome tetramers.

The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos.

1. Ribosomes and the tetramer arrangement peculiar to the tissues of chick embryos exposed to low temperatures were separated by sucrose-density-gradient centrifugation, and the effects of variation of the concentrations of Mg(2+), Ca(2+) and K(+) studied. 2. Lowering of the Mg(2+) concentration from standard buffer conditions caused a reversible dissociation of tetramers into monomers and of these into subunits. 3. Ca(2+) replaced Mg(2+) in causing the re-formation of tetramers and monomers from subunits after dissociation in low Mg(2+) concentrations. 4. Ca(2+) also caused an almost complete conversion of monomers into dimers in the presence of Mg(2+). 5. The effect of Ca(2+) on the formation of dimers was abolished by pretreatment of the ribosomes with ribonuclease, but the re-formation of tetramers was unaffected. 6. Increase of the K(+) concentration from that of the standard buffer caused dissociation of monomers and dimers into subunits. 7. Raised K(+) concentration also caused a stepwise alteration of the tetramer from a particle with a sedimentation coefficient of 197S, which constitutes the bulk of the tetramer at low K(+) concentrations, first to a 184S peak and finally to material with a sedimentation coefficient of about 155S. 8. The implications of these results on hypotheses of the arrangement of the individual monomers in the tetramer are discussed and a new model for the structure is proposed.

Ribosome tetramers from cold-treated chick embryos

Conference Article| April 01 1970 Ribosome tetramers from cold-treated chick embryos N. H. Carey N. H. Carey 1Searle Research Laboratories, Lane End Road, High Wycombe, Bucks., U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1970) 117 (3): 64P. https://doi.org/10.1042/bj1170064Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation N. H. Carey; Ribosome tetramers from cold-treated chick embryos. Biochem J 1 April 1970; 117 (3): 64P. doi: https://doi.org/10.1042/bj1170064Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1970 The Biochemical Society1970 Article PDF first page preview Close Modal You do not currently have access to this content.