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

Abstract

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. Binding of alkali-metal cations to RNA and some other associations were observed in such solvent systems. Possible effect of ionic association on mobilities are discussed. Rabbit-reticulocyte ribosomes were subjected to electro-phoresis as above, after their nascent-protein moiety had been labelled with [14C]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. 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. 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.