scholarly journals RIBOSOME CRYSTALLIZATION IN CHICKEN EMBRYOS

1972 ◽  
Vol 52 (2) ◽  
pp. 338-354 ◽  
Author(s):  
T. Morimoto ◽  
G. Blobel ◽  
D. D. Sabatini
Keyword(s):  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Ribosomal Subunits ◽  
Chicken Embryos ◽  
Polyuridylic Acid ◽  
Insoluble Product ◽  
Rnase Treatment ◽  
Low Ionic Strength ◽  
Polypeptide Chains

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 MgCl2 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-3H 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.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

scholarly journals Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells

1981 ◽  
Vol 194 (3) ◽  
pp. 1007-1009 ◽  
Author(s):  
J Schuck ◽  
G Reichert ◽  
O G Issinger
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Ribosomal Proteins ◽  
Ribosomal Subunits ◽  
Phosphorylated Amino Acid ◽  
Mouse Ascites ◽  
Independent Protein ◽  
Ascites Tumour Cells

Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro.

scholarly journals THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

1972 ◽  
Vol 54 (1) ◽  
pp. 56-74 ◽  
Author(s):  
Paul M. Lizardi ◽  
David J. L. Luck
Keyword(s):  
Protein Synthesis ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Selective Inhibitor ◽  
Mitochondrial Protein Synthesis ◽  
Ribosomal Subunits

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 130-145 ◽  
Author(s):  
G. Blobel ◽  
D. D. Sabatini
Keyword(s):  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Subunit Structure ◽  
Amino Acid Residues ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Cell Fractions ◽  
Carboxy Terminal

Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ∼39 amino acid residues, as estimated by chromatography on Sephadex G-50.

scholarly journals Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts.

1975 ◽  
Vol 65 (3) ◽  
pp. 513-528 ◽  
Author(s):  
M A Lande ◽  
M Adesnik ◽  
M Sumida ◽  
Y Tashiro ◽  
D D Sabatini
Keyword(s):  
Messenger Rna ◽  
Membrane Fraction ◽  
Human Diploid Fibroblasts ◽  
Intact Membrane ◽  
Direct Association ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
High Salt Buffer ◽  
Rnase Treatment ◽  
Polypeptide Chains

Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.

scholarly journals RIBOSOME-MEMBRANE INTERACTION

1973 ◽  
Vol 56 (1) ◽  
pp. 206-229 ◽  
Author(s):  
M. R. Adelman ◽  
David D. Sabatini ◽  
Günter Blobel
Keyword(s):  
Ionic Strength ◽  
Room Temperature ◽  
Ribosomal Subunit ◽  
High Ionic Strength ◽  
Ribosomal Subunits ◽  
Insoluble Material ◽  
Polyuridylic Acid ◽  
Nascent Chain ◽  
Membrane Bound ◽  
Polypeptide Chains

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl2, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [3H]puromycin into hot acid-insoluble material and from the release of [3H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (∼15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.

scholarly journals Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation.

1976 ◽  
Vol 71 (1) ◽  
pp. 307-313 ◽  
Author(s):  
M Adesnik ◽  
M Lande ◽  
T Martin ◽  
D D Sabatini
Keyword(s):  
Messenger Rna ◽  
Membrane Fraction ◽  
Human Fibroblasts ◽  
Ribosomal Subunits ◽  
Run Off ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Polypeptide Chains ◽  
Extensive Release

Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains.

scholarly journals Biogenesis of membrane-bound and secreted immunoglobulins. II. Two forms of the human alpha chain translated in vitro and processed in vivo as distinct polypeptide chains.

1981 ◽  
Vol 153 (6) ◽  
pp. 1684-1689 ◽  
Author(s):  
J M McCune ◽  
S M Fu ◽  
G Blobel ◽  
H G Kunkel
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Messenger Rna ◽  
Secretory Iga ◽  
Free System ◽  
Alpha Chain ◽  
Functional Consequences ◽  
Polypeptide Chains

Structural differences between alpha m (ther heavy chain of membrane IgA) and alpha s (the heavy chain of secretory IgA) were investigated. Messenger RNA from the human B lymphoblastoid line 32a.1, expressing both membrane and secretory IgA, was translated in a wheat germ cell-free system, resulting in the synthesis of two primary translation products for the alpha chain, that differed in molecular weight. In vivo pulse and pulse-chase experiments demonstrated that two early biosynthetic forms of the alpha chain were subsequently modified to yield three intracellular forms. As shown by endo-beta-N-acetylglucosaminidase H (endo H) treatment, these forms represent two alpha polypeptide chains, with varying compositions of N-linked oligosaccharides. Of the two forms of the alpha chain remaining after endo H treatment, only the form with the lowest molecular weight was associated with cells after long chase periods. The possible significance of this difference from the results with mu and delta chains is discussed. These results indicate that alpha m is distinguished from an alpha s by a difference in both primary structure and intracellular processing. The functional consequences of this distinction, previously shown for the heavy chain of membrane IgM (micrometer) and heavy chain of secretory IgM (microseconds), may reflect a principle common to the secretory and membrane forms of all immunoglobulin heavy chain classes.

scholarly journals eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

2021 ◽  
Author(s):  
Christopher P. Lapointe ◽  
Rosslyn Grosely ◽  
Masaaki Sokabe ◽  
Carlos Alvarado ◽  
Jinfan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Translation Initiation ◽  
Messenger Rna ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Architectural Framework ◽  
Subunit Joining ◽  
Structural Methods

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

Sign in / Sign up

Export Citation Format

Share Document