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A search for O-polypeptidyl-ribonucleic acids in rabbit-reticulocyte ribosomes by electrophoresis in phenol-acetic acid-water systems.

ABSTRACT: 1. When solutions of ;soluble' or transfer RNA (s-RNA) and of cytochrome c in phenol-acetic acid-water were mixed, intractable coacervates were formed. The addition to the solutions of various strong electrolytes prevented coacervation and allowed electrophoretic separation on filter paper. Protein migrates cationically, leaving RNA at or near the origin. The separation is aided by adsorption of RNA to the paper. Special arrangements were necessary to prevent contamination of the paper by ultraviolet-absorbing electrode-reaction products. 2. Binding of alkali-metal cations to RNA and some other associations were observed in such solvent systems. Possible effects of ionic association on mobilities are discussed. 3. Rabbit-reticulocyte ribosomes were subjected to electrophoresis as above, after their nascent-protein moiety had been labelled with [(14)C]valine in the intact cell. Most of the radioactivity migrated with the ribosomal protein; such protein as remained near the origin with the RNA had valine of lower specific radioactivity. 4. Molecular-sieve chromatography in phenol-acetic acid-water indicated that the nascent-protein moiety was not of markedly lower molecular weight than the average for the ribosomal proteins. 5. These results are very tentatively taken to mean that the nascent-protein moiety of ribosomes so prepared is not O-polypeptidyl-s-RNA. It is postulated that, in the course of adding each amino acid residue, the growing polypeptide chain is transferred from ester linkage with s-RNA to linkage with ribosomal protein through its carboxyl group, perhaps by ester linkage to an alcoholic group. The two types of intermediate, O-polypeptidyl-s-RNA and polypeptidyl-protein, would be found in different proportions, depending on the isolation procedure used. Some implications and possible tests of this hypothesis are discussed.

SUBMITTER: Brattsten I

PROVIDER: S-EPMC1264746 | biostudies-other | 1965 Dec

REPOSITORIES: biostudies-other

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Project description:1. The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.

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A search for O-polypeptidyl-ribonucleic acids in rabbit-reticulocyte ribosomes by electrophoresis in phenol-acetic acid-water systems.

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