Presence Of Ribosomes Research Articles

A proposed mechanism for ADP ribosylation of aminoacyl transferase II by diphtheria toxin.

Diphtheria toxin inhibits amino acid incorporation into proteins in cell-free, mammalian systems (Collier and Pappenheimer, 1964; Goor and Pappenheimer, 1967; Goor et al., 1967) by inactivating aminoacyl transferase II (Collier, 1967). In addition, toxin also inhibits the following activities of aminoacyl transferase II (T2): 1. ribosomal-dependent GTPase activity of T2 (Raeburn et al., 1968; Honjo, Nishizuka, and Hayaishi, this volume); 2. translocation of nascent peptide chains as measured by the T2-dependent increase in the number of chains reactive with puromycin (Schneider et al., 1968); 3. the binding of GTP to T2 in the presence of ribosomes (Raeburn et al., 1968).

Protein synthesis during fungal spore germination. II. Aminoacyl-soluble ribonucleic acid synthetase activities during germination of Botryodiplodia theobromae spores.

The specific activities of 13 aminoacyl-soluble ribonucleic acid (sRNA) synthetases were measured at various time intervals during the germination of Botryodiplodia theobromae conidiospores. The enzyme activities were low or absent in ungerminated spores, and they increased rapidly as germination proceeded. When extracts of the ungerminated spores were prepared with mortar and pestle, very little or no enzyme activity was detected. When the extracts were prepared with a mechanical homogenizer, however, they exhibited some enzyme activity, although less than did the extracts from germinated spores. Enzyme activities from germinated spores were approximately the same, regardless of the method of preparation. The enzyme fraction from ungerminated spores prepared with a mechanical homogenizer could also stimulate incorporation of phenylalanine into polyphenylalanine in the presence of ribosomes, polyuridylic acid, and sRNA, although the activity was approximately only 15 to 20% that of a similar enzyme fraction from germinated spores. It is concluded that ungerminated spores of B. theobromae contain active aminoacyl-sRNA synthetases and transfer enzymes, although the activities are low when compared to germinated spores.

Studies on human platelets. I. Synthesis of platelet protein in a cell-free system

A reproducible cell-free system, active in protein synthesis, has been prepared from human blood platelets. The ability of this system to incorporate 14C-labeled amino acids, is dependent on the presence of ribosomes, a soluble enzyme fraction (S-150), and ATP plus an ATP-generating system. Both puromycin and ribonuclease have the ability to inhibit amino acid incorporation. A major soluble protein product of this system has been characterized; the solubility properties, sucrose gradient patterns, and fingerprint patterns of the 14C-labeled soluble product appear identical to the [ 14C]actomyosin-like contractile protein isolated from labeled, intact platelets.

Reaction of Ribosome-bound Peptidyl Transfer Ribonucleic Acid with Aminoacyl Transfer Ribonucleic Acid or Puromycin

Abstract Polylysyl transfer ribonucleic acid, free of lysyl-tRNA, has been isolated and its characteristics in an Escherichia coli polypeptide-synthesizing system in vitro have been studied. Ribosomes obtained from frozen E. coli cells were washed in 0.5 m NH4Cl and assayed at MgCl2 concentrations of at least 0.01 m. Binding of this peptidyl-tRNA to ribosomes occurs in the presence of templates containing 3 or more adenylic acid residues. p-Chloromercuriphenylsulfonate or lysyl-tRNA prevents this attachment. Binding occurs most effectively when both 30 S and 50 S ribosomal subparticles are present. Unlike lysyl-tRNA, bound polylysyl-tRNA reacts with puromycin in the absence of added GTP and supernatant factors. The product of this reaction was shown to be polylysylpuromycin. Under analogous conditions, a limited incorporation of lysine into bound polylysyl-tRNA can be shown. In this limited addition reaction, a polyadenylate template longer than three nucleotides is required. Chlortetracycline is a strong inhibitor of the limited addition of lysine to polylysyl-tRNA, but is without effect on the cleavage of polylysyl-tRNA by puromycin. Chloramphenicol, on the other hand, inhibits the reaction between polylysyl-tRNA and puromycin, but has little influence on the limited addition reaction. Gougerotin, a puromycin-like antibiotic, inhibits the puromycin reaction. Phenylalanyl-tRNA reacts poorly with puromycin in the presence of ribosomes and oligouridylate, whereas N-acetylphenylalanyl-tRNA is cleaved extensively, forming N-acetylphenylalanylpuromycin. Phenyllactyl-tRNA, although capable of binding to ribosomes, displays no detectable reactivity with puromycin. Chloramphenicol completely inhibits the cleavage of N-acetylphenylalanyl-tRNA by puromycin.

Inhibitors of the reaction between puromycin and polylysyl-RNA in the presence of ribosomes

Inhibitors of the reaction between puromycin and polylysyl-RNA in the presence of ribosomes

Effect of ribosomes on the biosynthesis of ribonucleic acid in vitro

A deoxyribonucleoprotein that contains RNA polymerase activity and DNA template has been isolated from Escherichia coli and partially purified by zone sedimentation. This preparation synthesizes RNA in the presence of all four nucleoside triphosphates and in the absence of exogenous DNA; the nascent RNA remains attached to the enzyme—template complex during the polymerization reaction. The addition of ribosomes stimulates the synthesis of RNA; the RNA made in the presence of ribosomes is of higher molecular weight than that made in their absence and a significant portion of the nascent RNA is released from the enzyme—template complex, in the form of RNA-ribosome aggregates. Evidence has been obtained that ribosomes, natural acceptors for the nascent RNA, exert a direct effect on RNA synthesis as a consequence of attachment to the growing chain, and possibly of ribosome movement, but not of protein synthesis-dependent movement of the ribosome along the RNA chain.

Interchangeability of ribosomes and protein synthesizing enzymes from Penicillium cyclopium and those prepared from other organisms.

IT has been established that the incorporation of amino-acids into peptide chains in vitro requires the presence of ribosomes, soluble ribonucleic acid (sRNA), soluble enzymes (105,000g supernatant enzymes) and messenger ribonucleic acid. The number of reports on the interchangeability of ribosomes prepared from one organism and soluble enzymes prepared from another are, however, limited1–5. In addition, there are only two reports on in vitro amino-acid incorporating systems developed from mycelial fungi6,7. This note briefly reports on the preparation of an in vitro amino-acid incorporating system from the mycelial fungus, Penicillium cyclopium, and the interchangeability of its ribosomes and supernatant enzymes with those prepared from a bacterium, Escherichia coli, a yeast, Saccharomyces cerevisiae, and a higher plant, seedlings of castor bean (Ricinus communis).

THE DEVELOPMENT OF CHLOROPLASTS AND PHOTOSYNTHETIC ACTIVITIES IN YOUNG BARLEY LEAVES

SummaryCarbon dioxide exchanges in the dark and in the light indicate that photosynthetic activity occurs m young leaves of barley seedlings after about 3 hours illumination under favourable conditions. Activity increases rapidly as chlorophyll is formed in the expanding leaf blade; a compensation point is reached after 30–40 hours and thereafter carbon dioxide assimilation exceeds respiration.Changes in the fine structure of plastids in the young leaves were examined by electron microscopy; some of the chief stages in the development of the lamella system and the differentiation of grana are described. The presence of ribosome‐ and nucleoplasm‐like structures in the chloroplast stroma was indicated.The relationship between the development of chloroplasts and their photosynthetic activity is discussed. It is suggested that the formation of distinctive double membranes between the thylakoids of the grana plays an important part in the structural organization of the pigment system. The action of light on chloroplast development indicates that a primary step in photomorphogenesis is the conversion of protochlorophyllide to chlorophyll. The further extension and development of the lamellar system probably depends upon the biosynthesis of proteins and lipids, closely linked with photosynthesis and operating under the control of nucleic acids.

Release of lysine peptides by puromycin from polylysyl-transfer ribonucleic acid in the presence of ribosomes

Release of lysine peptides by puromycin from polylysyl-transfer ribonucleic acid in the presence of ribosomes

Rapidly synthesized ribonucleic acid in membrane ghosts from Streptococcus fecalis protoplasts

1. 1. Small amounts of RNA are present in washed membrane ghosts prepared by lysis of Streptococcus fecalis (ATCC No. 9790) protoplasts. Evidence was sought for differences between the membrane ghost RNA and RNA in other cell fractions, particularly ribosomes. 2. 2. After pulse labelling intact cells with [ 32P]orthophosphate, the membrane ghost RNA was found to be the most rapidly labelled as judged by measurement of the specific radioactivity of isolated 32P-labelled 2′(3′)-mononucleotides. Insoluble, easily sedimentable fragments of membrane ghosts obtained after sonication still retained RNA also with the most heavily 32P-labelled nucleotides. The nucleotide composition of the bulk and newly synthesized [ 32P]RNA in the membrane ghosts resembled the composition of ribosome RNA and the RNA in a DNA-rich fraction, but not s-RNA. 3. 3. Electron micrographs of the membrane ghosts indicate the presence of ribosomes. They are associated with a filamentous network and they are also situated in large numbers within a hollow tubular structure. 4. 4. It is concluded that in Streptococcus fecalis a rapidly synthesized RNA is associated specifically with membranous structures probably in membrane-associated ribosomes. 5. 5. Some information on the nature and amounts of other chemical components in the membrane ghosts is presented.

INFLUENCE OF TESTOSTERONE AND POLYURIDYLIC ACID ON THE INCORPORATION OF PHENYLALANINE INTO PEPTIDE LINKAGE BY PROSTATIC RIBOSOMES.

RECENT studies1 on the transfer of radioactivity from soluble ribonucleic acid-(sRNA)-valine-14C to protein-like material by isolated prostatic ribosomes suggested that the levels of template RNA associated with these particles are controlled by testicular hormones. In line with this conclusion was the observation that randomly ordered copolymers of uridylic and guanylic acids (poly UG) stimulated valine incorporation by prostatic ribosomes from orchiectomized animals to a much greater extent than was found with ribosomes prepared from castrates which were treated with testosterone. However, two considerations complicate interpretation of the response of cell-free valine incorporating systems to poly UG. First, poly UG (prepared by the action of polynucleotide phosphorylase on a mixture of uridine and guanosine diphosphates) represents a complex mixture of polyribonucleotides of varying size and base sequences, the exact composition of which is difficult to ascertain. Secondly, the effects of guanine-containing polyribonucleotides on ribosomal amino-acid incorporating systems seem to depend not only on the base compositions of the polymers, but also on the degree of ordered secondary structure which they may exhibit in solution2. It became of interest, therefore, to examine the response of prostatic ribosomes to polyribonucleotides containing only one base. It is well established that polyuridylic acid (poly U) directs the entry of phenylalanine into peptide linkage in the presence of ribosomes from a number of tissues3–8. The following experiments concern factors which influence the transfer of radioisotope from sRNA-phenylalanine-14C to acid-insoluble products by prostatic ribosomes, incubated in the absence and presence of poly U.

Studies on the incorporation of amino acids in a yeast cell-free system

1. 1. Some properties of the system for the incorporation of amino acids into the cell-free preparation have been studied. The system requires a supply of ATP (and its generating system), and also the presence of ribosomes. 2. 2. s-RNA has been found to be an essential intermediate in the incorporation of amino acids into protein, but the RNA component in the ribosomal fraction also takes part. 3. 3. Chloramphenicol, DNAase (EC 3.1.4.5), or the supply of all four ribonucleoside triphosphates has no marked effect on the incorporation, but puromycin and RNAase (EC 2.7.7.16) are potent inhibitors. 4. 4. The nature of the protein synthesized in this system has been examined using chromatography on DEAE-cellulose columns. Radioactivity was observed only in the protein of the ribosomal fraction and alkali-eluting part, and not in the soluble protein portion. 5. Using these results, the characteristics of the present incorporation system were discussed.