Unstable Ribonucleic Acid Revealed by Pulse Labelling of Escherichia Coli

Gros, François; Hiatt, Howard H.; Gilbert, Walter; Kurland, C. G.; Risebrough, Robert W.; Watson, James D.

doi:10.1038/190581a0

Cited by 591 publications

(140 citation statements)

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“…Bacterial ribosomes can be dissociated readily into two subparticles and messenger RNA by reducing the magnesium concentration of the medium [1,2], but mammalian ribosomes appear to be more stable. The conditions under which they can be dissociated have been investigated by a number of workers who have shown that treatment with EDTA [3] or pyrophosphate [4], or exposure to high pH [5] is required.…”

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The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

Nolan

Arnstein

1969

European Journal of Biochemistry

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Rabbit reticulocyte ribosomes were incubated with polyuridylic acid a t 37" for 1 h under conditions suitable for protein synthesis. Ribosomes containing bound polyuridylic acid were isolated and dissociated into subparticles by treatment with EDTA. The subparticles were separated by zone centrifugat,ion in a sucrose density gradient and the distribution of polyuridylic acid was examined by measuring the ability of gradient fractions to stimulate the incorporation of [14C]phenylalanine into protein by a cell-free system. It was observed that the peak of polyuridylic acid activity corresponded to a region of the subparticle gradient having a sedimentation coefficient of about 50 S. When [14C]polyuridylic acid was used in the initial binding reaction, subsequent analysis of subparticles produced by treatment with EDTA confirmed the presence of polyuridylic acid on or close t o the large ribosomal subparticle.Bacterial ribosomes can be dissociated readily into two subparticles and messenger RNA by reducing the magnesium concentration of the medium [1,2], but mammalian ribosomes appear to be more stable. The conditions under which they can be dissociated have been investigated by a number of workers who have shown that treatment with EDTA [3] or pyrophosphate [4], or exposure to high pH [5] is required.The fate of mRNA when mammalian ribosomes are dissociated into subparticles is uncertain. On the one hand, Huez et al. [6] reported that treatment of reticulocyte polysomes with EDTA releases a minor RNA component which is rapidly labelled in vivo and sediments with a coefficient of 9-10 S. On the basis of sue, kinetics of labelling and other properties this RNA is considered to be haemoglobin messenger [7], but it should be noted that it has not yet been shown to have any stimulatory activity for cell-free protein synthesis. On the other hand, stimulatory RNA was recovered in the ribosomal subparticles after dissociation of reticulocyte ribosomes, most of the stimulatory activity being associated with the RNA from the large (60 S) subparticle [5]. The validity of using stimulation of cell-free protein synthesis as a criterion of messcnger activity has recently been questioned by Hunt and Wilkinson[8] who showed that the synthesis of a haemoglobin-like product by a cell-free preparation from reticulocytes was stimulated by liver as well as by reticulocyte RKA. It is possible, therefore, that cell-free protein I n view of these uncertainties, we decided to investigate the fate of a synthetic messenger, poly U, after interaction with reticulocyte ribosomes in a cell-free protein-synthesizing system and subsequent dissociation of the ribosome-poly U complex into subparticles by treatment with EDTA. This approach has the advantage that the messenger can be identified readily, either by its ability to synthesize a characteristic polypeptide or by interacting ribosomes with radioactive poly U. Both methods were used in the present experiments, which lead to the conclusion that a substantial part of the poly U is not rel...

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The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

Nolan

Arnstein

1969

European Journal of Biochemistry

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“…Bacterial ribosomes have been readily dissociated into active subunits by lowering the magnesium ion concentration (7,23,25). With mammalian ribosomes (polyribosomes preparation), incubation in a high concentration of monovalent cation in the absence of MgCl2 has only resulted in a decrease in the proportion of polyribosomes (3), while complete dissociation into subunits required a high pH or a chelating agent such as EDTA (6).…”

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Dissociation of N₂ Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

Lin

Key

1971

Plant Physiol.

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The dissociation of N2 gas-induced monomeric ribosomes from the pea root was studied by varying the concentration of KCI (or NILCI) and MgCL2 in the presence of dithiothreitoL These monoribosomes were shown to dissociate completely into subunits at 0.5 M KCI or NH4CI in the presence of 5 mM MgCI2. The 40S subunits were more susceptible to structural change in KC1 than were the 60S subunits. On the other hand, the 60S subunits appeared to be more labile to NH4CI.The activity of the subunits relative to aminoacyl-tRNA binding and peptide bond formation was investigated using subunits derived from 0.5 M KCI (or NH4CI)

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“…A part of the rapidly labeled polysomal RNA can be reversibly separated from rRNP by the action of EDTA (1.25 to 5 pmoles EDTA/mg RNP). It sediments in a broad zone (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) S) of high specific radioactivity. Increasing Mg++ concentration results in the reassociation of rapidly-labeled RNA to ribosomal sub-particles.…”

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Acides ribonucléiques messagers de la glande thyroïde

Cartouzou

Attali

Lissitzky

1968

European Journal of Biochemistry

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( R e p le 2 aoDt 1967/6 dkcembre 1967) I . Nuclear and cytoplasmic RNA from sheep thyroid slices in vitro labeled with [3H]uridine or [32P]phosphate from 5 min up to 9 h, have been obtained by different phenol extraction procedures. Early-labeled RNA was only extracted by phenol containing 0.1 or lo/, (w/v) sodium dodecylsulfate (SDS) at 0" or 60°, whereas stable nuclear or polysomal RNA was easily extracted by phenol at 0".2. Time course of incorporation of label into subcellular particles has shown the early labeling of nuclear RNA and the transfer of radioactivity into the cytoplasm.3. Rapidly-labeled RNA extracted from nuclei by phenol-I O/, (w/v) SDS and analyzed by sedimentation was shown to sediment in three discrete classes 50-30 S, 22 S and 13 S, different from ribosomal RNA. After 9 h o f incubation the 22 S and 13 S labeled RNAs were still present in detectable amounts in the nuclei, indicating a somewhat low turnover rate for these components. 4.The kinetics of incorporation of label into polysomes was studied in thyroid slices incubated from 15 min up to 5 h with [3H]uridine or [32P]phosphate. RNA was dissociated from polysomes by dodecylsulfate treatment and centrifuged on sucrose gradient. As for nuclei, the rapidlylabeled RNAs sediment in discrete size classes, 22 S and 13 S. After a long incubation time (5 h) the label was predominant in 28 S and 18 S ribosomal RNA but 28 S RNA was not completely labeled. Up to 30 min labeling a high molecular weight, early-labeled RNA was present in purified polysomes. Its radioactivity declines after 60 min labeling.5. A part of the rapidly labeled polysomal RNA can be reversibly separated from rRNP by the action of EDTA (1.25 to 5 pmoles EDTA/mg RNP). It sediments in a broad zone (12-30 S) of high specific radioactivity. Increasing Mg++ concentration results in the reassociation of rapidly-labeled RNA to ribosomal sub-particles.6. Buoyant densities of the polysomal RNP particles obtained by EDTA treatment of 32P-labeled polysomes and stabilized by means of formaldehyde fixation, were determined by banding in CsCl gradients. The bulk o f the stable material banded at characteristic densities (d = 1.55 and 1.48), but most of the rapidly-labeled RNA banded at d = 1.38.The specific radioactivity of this material was the same as that of the 12-30 S RNA isolated by sucrose gradient centrifugation from the same EDTA-treated polysomes. Preliminary double labeling experiments and computation of the RNA to protein ratio calculated from the RNA and protein density values indicate this material to be early-labeled nucleoprotein particles with a high protein content.La glande thyroide est un tissu dont la fonction essentielle est la synthhse des hormones thyroidiennes. Morphologiquement la d86renciation du tissu thyroidien se manifeste par une structure folliculaire trhs caractkristique. Chaque follicule est constitu6 [12] le poids moleculaire des sousunites obtenues par dissociation de la thyroglobuline reduite et 8-carboxymethylee serait de 110.000 B 125.000, indiquan...

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Unstable Ribonucleic Acid Revealed by Pulse Labelling of Escherichia Coli

Cited by 591 publications

References 14 publications

The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

Dissociation of N₂ Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

Acides ribonucléiques messagers de la glande thyroïde

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Unstable Ribonucleic Acid Revealed by Pulse Labelling of Escherichia Coli

Cited by 591 publications

References 14 publications

The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid

Dissociation of N2 Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

Acides ribonucléiques messagers de la glande thyroïde

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Dissociation of N₂ Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea