The growth of ordered two-dimensional sheets of 70 S ribosomes from Bacillus stearothermophilus

Well ordered two-dimensional sheets of intact 70 S ribosomes from Bacillus stearothermophilus have been obtained in vitro using salt-alcohol mixtures. These sheets consist of relatively small unit cells with dimensions of 200 +/- 20 A and 400 +/- 30 A. Diffraction patterns of electron micrographs of these sheets stained with uranyl acetate contain features to 42 A resolution.     

Approaching the molecular structure of ribosomes

Fifteen forms of three-dimensional crystals and three forms of two-dimensional sheets from ribosomal particles have been grown. In all cases only biologically active particles could be crystallized, the crystalline material retaining its integrity and biological activity for months. Cryastallographic data have been collected from crystals of 50 S ribosomal subunits, using synchrotron radiation, at temperatures between 19 and -180 degree C. Although at around 0 degrees C in the synchrotron X-ray beam the crystals rapidly lose their high-resolution reflections, at cryo-temperatures hardly any radiation damage occurs over long periods, and a complete set of diffraction data to about 6 A resolution could be collected from a single crystal. Heavy-atom clusters were used for soaking as well as for specific binding to the surface of the ribosomal subunits prior to crystallization. The 50 S ribosomal subunits from a mutant of Bacillus stearothermophilus which lacks the ribosomal protein BL11 crystallize isomorphously with the native form. Models of the entire 70 S ribosome and of the 50 S subunit have been reconstructed from two-dimensional sheets at 47 and 30 A, respectively. These models demonstrate the overall shape of the particles, the contact areas between large and small subunits, the space where protein biosynthesis may take place and a tunnel through the 50 S subunit which could provide a path for the nascent polypeptide chain.     

Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2

Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays.     

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal subunits containing a nascent polypeptide chain

Polylysine chains were synthesized on Bacillus stearothermophilus ribosomes in a poly(A)-programmed in vitro system. After separation of the ribosomal subunits by sucrose gradient centrifugation, the polylysine chains (in contrast to the polyphenylalanine chains synthesized in a poly(U) system) reproducibly remained attached to the large ribosomal subunit. It was possible to produce two-dimensional crystalline sheets from the large ribosomal subunits containing the polylysine chains. These sheets are an essential prerequisite for three-dimensional reconstruction studies aiming to show that the tunnel in the large ribosomal subunit provides a path for the nascent polypeptide chain.     

Miniature two-dimensional thin-layer chromatographic separation of lecithin and sphingomyelin

A miniature two-dimensional thin-layer chromatographic procedure employing silica gel impregnated glass-microfiber chromatography sheets (commercial product, ITLC-type SG sheets) has been developed for the separation of lecithin (L) and sphingomyelin (S) from a standard lipid mixture containing L, S, lysolecithin, phosphatidyl inositol (PI), phosphatidyl serine (PS), phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The newly developed procedure eliminates possible interference from PI and PS. Complete separation of L and S was easily achieved with chromatographic solvent migration times of approximately 3 and 2 min for the first and second dimensions, respectively. The lipids were visualized by charring and fluorescent staining techniques. The procedure has been adapted for the separation of L and S from amniotic fluid samples.     

Three-dimensional image reconstruction from ordered arrays of 70S ribosomes

A better understanding of the molecular mechanism of protein biosynthesis still awaits a reliable model for the ribosomal particle. We describe here the application of a diffraction technique, namely three-dimensional image reconstruction from two-dimensional sheets of 70S ribosomes from Bacillus stearothermophilus at 47 A resolution. The three-dimensional model obtained by these studies shows clearly the two subunits, the contact points between them, an empty space large enough to accommodate the components of protein biosynthesis, the location of regions rich in RNA and a possible binding site for mRNA. The tunnel within the 50S particle which may provide the path taken by the nascent polypeptide chain in partially resolved.     

Dissociation of yeast 80 S ribosomes by chaotropic salts

Exposure of yeast 80 S ribosomes to chaotropic salts such as NaClO₄ or NaSCN at concentrations as low as 0.4 M resulted in complete dissociation and subsequent aggregation of the ribosomal proteins. However, under similar conditions, both NaCl and NaBr did not cause dissociation and aggregation. The protein precipitate obtained by exposing the ribosomes to 0.5 M NaClO₄ was free of any rRNA contamination as judged by ultraviolet-absorption analysis. Comparison of the two-dimensional polyacrylamide gel electrophoretic analysis of the above ribosomal protein precipitate with that ribosomal proteins isolated by the standard acetic acid extraction procedure revealed that the protein precipitate contained all the ribosomal proteins. Based on these results, a simple method for the isolation of total ribosomal proteins and rRNA under mild, nondenaturing conditions is proposed. A possible mechanism for the dissociation of proteins from the ribosome by chaotropic salts is also discussed.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Isolation, characterization and restriction endonuclease mapping of the Petunia hybrida chloroplast DNA

A procedure is developed for the isolation of intact chloroplast DNA (ctDNA) from Petunia hybrida. The molecular weight, calculated from contour length measurements, is 96.0 +/- 4.5 x 10(6) daltons. This value is in good agreement with the value of 101.2 x 10(6) daltons that was estimated from the electrophoretic mobilities of restriction endonuclease fragments of ctDNA. Analysis of petunia ctDNA in neutral CsCl gradients revealed the presence of only one type of DNA at a buoyant density of 1.6987 +/- 0.0005 gcm-3. This corresponds with a GC-content of 39.3 +/- 0.5%. A physical map of petunia ctDNA was constructed by using the restriction endonucleases Sal I, Bgl I, Hpa I and Kpn I. The map indicates that petunia ctDNA contains two copies of a sequence in an inverted orientation. The inverted repeat regions have a minimum length of 10 x 10(6) daltons. Hybridization data indicate that part of the inverted repeat regions contain the genes for chloroplast ribosomal RNAs.     

Two-dimensional restriction analysis of the Bacillus subtilis genome: gene purification and ribosomal ribonucleic acid gene organization.

With two-dimensional restriction enzyme analysis we have been able to cleave the Bacillus subtilis genome and resolve the resulting deoxyribonucleic acid (DNA) segments into discrete bands on agarose gels. A general procedure for gene purification has been developed by coupling multidimensional restriction analysis with a biological assay for gene detection. The organization of ribosomal ribonucleic acid (rRNA) genes was studied by hybridizing 16S and 23S rRNA probes to the two-dimensional DNA banding patterns.     

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.     

An improved direct RNA sequence method; its application to Vicia faba 5.8S ribosomal RNA.

We have developed a direct read-off sequencing procedure, based on the method of Stanley and Vassilenko using E. coli 5S ribosomal RNA as a model compound. Radioactive bands were transferred from an acrylamide gel fractionation in the first dimension onto a DEAE-cellulose thin layer plate. After in situ enzymatic digestion with RNase T2, mononucleoside 3',5'-diphosphates were separated in the second dimension by electrophoresis at pH 2.3. Using this two-dimensional procedure the entire sequence of 163 residues of the previously unknown Vicia faba (broad bean) 5.8S ribosomal RNA was deduced.     

The Staphylococcus aureus alpha-toxin channel complex and the effect of Ca2+ ions on its interaction with lipid layers.

Using the techniques of two-dimensional crystallization on supported lipid bilayers together with computer image processing, two distinct two-dimensional crystal types of staphylococcal alpha-toxin complex are formed depending on the presence or absence of Ca2+ ions. Without Ca2+, these are hexagonally packed (in A, a = b = 89.5 +/- 2.5 A; theta = 119.7 degrees) With Ca2+ present, rectangular crystal packing is seen (in A, a = 114.8 +/- 1.6 A, b = 140.2 +/- 0.7 A; theta = 89.1 degrees). A third, banded crystal type is also seen which is interpreted as a side-to-side packing of regular tubules. We use these tubular crystals for cross-correlation searches with top and side-on views of the complex from single particle reconstructions, and with the repeating units from the two-dimensional crystal types. The results lead us to propose a model in which the different two-dimensional crystal types are formed as a result of alpha-toxin hexamers packing in different orientations. In the hexagonal crystals the hexamers lie end-on with a 6-fold axis in projection. On the addition of Ca2+, the hexamers reorient to lie tilted with respect to the support, thus giving rise to a rectangular projection.     

Cold shock and its effect on ribosomes and thermal tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0 degrees C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 +/- 0.1 degrees C (mean +/- standard deviation) to 72.1 +/- 0.5 degrees C (P < or = 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 +/- 0.4 degrees C to 66.8 +/- 0.5 degrees C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.

Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.     

Estimation of lipid concentrations on thin-layer plates by densitometry of transparent copies

A relatively simple procedure for the quantitative estimation of phospholipids resolved on thin-layer plates has been developed. After resolution in an appropriate solvent, the lipids are visualized by staining with iodine, ninhydrin, or molybdate and then photocopied onto transparent sheets with a standard office copy machine. The density of each spot on the photocopy, measured with a simple silicon cell area densitometer, is a direct function of each lipid applied to the plate over at least a six- to eightfold range in concentration. Under controlled conditions the staining and photocopying steps are quite reproducible. Known concentrations of the choline, ethanolamine, serine, and inositol derivatives of L-alpha-phosphatidic acid applied either separately or as mixtures can be determined essentially quantitatively (100 +/- 5%) by this procedure following their resolution on the thin-layer plates.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

Oligonucleotide sequences of pancreatic and T1 ribonuclease digests of 5S ribosomal RNA from mouse cells.

Oligonucleotides produced by complete pancreatic and T1 RNase digestion of 5S ribosomal RNA from a mouse hepatoma, MH 134, have been separated with two-dimensional electrophoresis and their nucleotide sequences determined. Except for the presence of a 5'-terminal diphosphate, these nucleotide sequences were identical with those of KB cells, confirming the identity of the primary structure of 5S RNA between these animals. Both oligonucleotide patterns produced with these enzymes from 5S RNA of the liver were also identical with those of the hepatoma. All these agree with the strong conservation of 5S RNA genes in animal species. However, when 5S ribosomal RNA was extracted from ribosomes which were prepared from microsomal pellet, pancreatic RNase digest contained two trinucleotides (A-G-Cp and G-A-Cp) that were not found in 5S RNA prepared with a one-step procedure. It was concluded that different isolation procedure might indeed cause artifactual fragments on enzymatic digestion due to internal nicks produced during isolation. The significance of 5'-terminal diphosphate in relation to the biosynthesis of 5S ribosomal RNA is also discussed.     

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.     

Immunological comparison of individual ribosomal proteins in six species of the genus Podospora

Summary The ribosomal proteins of six species belonging to the genus Podospora have been compared by electrophoretical and immunological methods. The important variability in the electrophoretical properties is similar to other lower eukaryotes but we could specify the extent of the variability of the r-proteins by the analysis of immunological relationships between individual proteins. Radioimmuno-detection of ribosomal proteins blotted on nitrocellulose sheets after separation on two-dimensional gels, was used to determine common antigenic sites on the r-proteins of the different species. These sites may be present on proteins that do or do not have identical electrophoretical mobilities. Furthermore, the proteins which co-migrate do not necessarily cross-react.