Studies on the synthesis of ribonucleotide homopolymers

Previous work has shown that cytoplasmic fractions of animal cells are capable of the incorporation of ribonucleoside triphosphates into RNA. The present investigation was carried out to elucidate the mechanism and properties of such enzymes in rat liver cells. Female rats were anaesthetised, the abdomens opened, the livers perfused, and then removed. Enzyme fractions were prepared by differential centrifugation of liver homogenates. The incorporation of radioactive labelled ribonucleoside triphosphates into polyribonucleotides catalysed by such fractions was measured. The microsomal fraction was most active in incorporating (a-32P) UTP into polyribonucleotides. Such activity required a pH of 7.5-8.0 and the addition of Mg++ rather then Mn++ ions. The uptake of (a--32P) UTP was promoted presence of ATP, GTP and CTP, and an ATP generating system. While the addition of RNA stimulated the reaction and ribonuclease completely abolished the reaction, the addition of DNA, deoxy- ribonuclease or actinomycin had no effect on the uptake of (a-32P) UTP. It was concluded that incorporation of ribonucleoside triphosphates was by an RNA-, rather than a DNA- dependent reaction. Alkaline hydrolysis of the reaction products after (a-32P) UTP incorporation, end measurement of the radio-activity in the 2'(3') monophosphates so obtained, indicated that the main product was sequences of uridylic acid. It was not possible, however, to exclude hetero-polymer formation. All four ribonucleoside triphosphates were incorporated into acid insoluble products. In no case was maximum incorporation obtained in the presence of all four ribonucleoside triphosphates, reducing the probability of heteropolymer formation. The microsomes were fractionated into rough surfaced vesicles, smooth surfaced vesicles and free ribosomes. No activity was recovered from the smooth surfaced vesicle fraction. Activity in the rough surfaced vesicle fraction, appeared to be due to contamination by a supernatant fraction. An enzyme fraction with very high specific activity was recovered with the free ribosomes. Washing the ribosomes in dense sucrose solutions and isolation of the free ribosome fraction from media containing high concentrations of EDTA had no effect on the activity. It could be demonstrated unequivocally that the incorporation of UTP represented addition of seven to eight residues of uridylic acid to the 2'(3') ends of pre-existing RNA chains. ATP end CTP, but not GTP, were also incorporated into polyribonucleotides. Following the uptake of 3H-UTP, the nucleic acids were extracted from reaction mixtures, Using a combination of fractionation by sucrose gradient centrifugation and on columns of G-100 Sephadex, it was possible to exclude ribosomal RNA, 5S-RNA and transfer RNA as endogenous primers. This left the interesting possibility that messenger RNA acted as acceptors for the homopolymer sequences. The biological significance and the relationship of the free ribosomal enzyme(s) to other known enzymes of polynucleotide metabolism are discussed in some detail. The uptake of ribonucleoside triphosphates by a supernatant fraction was investigated. The uptake of UTP required. the presence of Mg++ ions and en added RNA primer. The incorporation of UTP could adequately be described as terminal addition of one to two ribonucleotides to the 2'(3') ends of pre-existing RNA primers. The enzyme (s) appeared to require free 2' (3') hydroxyl groups on the primer molecules. The significance of this activity is discussed