Studies on the nature of polysomes

Abstract

Conditions needed to achieve optimum amino acid incorporation into protein in vitro with rat-liver ribosomes, polysomes and a preparation of the total ribonucleoprotein particles are defined. Enough cell sap (from which inhibitors had been removed) must be added so that it is not limiting. The RNA strand holding ribosomes in the polysome structure had some of the characteristics of messenger RNA since it was rapidly labelled with [H3]orotic acid and its synthesis was sensitive to treatment of the rats with actinomycin. It was shown that a polysome structure was not essential for amino acid incorporation into protein and that single ribosomes, suitably attached to messenger RNA, could be active. Polysome aggregates could be degraded to smaller and ultimately mainly to monomeric and dimeric ribosomes by lowering the Mg2+ ion concentration of the suspending medium. This process was not caused by ribonuclease activity but by plucking ribosomes from the polysome structure and was reversible only to a very limited extent. Polysomes, centrifuged through media containing various magnesium concentrations, yielded varying proportions of different sizes of ribonucleoprotum particles, yet the ability of the preparations to incorporate amino acids into protein were not greatly different from one another. Polysomes prepared in the presence or absence of Mg, differed but slightly in their ability to incorporate amino acids into protein. Ribonucleoprotein particles from liver of a rat given an injection of radioactive amino acids were analyzed by sucrose-gradient centrifugation in the presence or absence of Mg2+ ions. The specific activity was higher in the heavy region and the fall of specific activity steeper in the absence of Mg2+ ions. A similar analysis after [H3] orotic acid labelling of the messenger RNA again showed a difference of the same type in the slopes of the gradients in the presence or absence of Mg2+ ions. Not each ribosome of the polysome structure is active and that the inactive particles are preferentially removed on decreasing the Mg concentratio.