Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes.

Abstract

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l‐[14C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg2+ for basal incorporation of amino acids into protein was 8 mm; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg2+. The response to nuclear RNA was also enhanced as the K+ concentration was increased from 25 to 100 mm. The stimulatory effect of nuclear RNA on incorporation of l‐[12C]eucine was either unaltered or depressed by addition of a mixture of 19 l‐[12C]amino acids each at concentrations, of 10−8, 10−2, or 10−1 mm. Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations.Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid‐acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid‐acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.