Reactions at the 3′ Terminus of Transfer Ribonucleic Acid: VI. PROPERTIES OF THE POLY(C) POLYMERASE ACTIVITY ASSOCIATED WITH RABBIT LIVER TRANSFER RIBONUCLEIC ACID NUCLEOTIDYLTRANSFERASE

Abstract

Abstract The properties of the poly(C) polymerase activity associated with rabbit liver tRNA nucleotidyltransferase were examined. The anomalous activity co-purified with tRNA nucleotidyltransferase throughout a 25,000-fold purification and was associated with both forms of the latter enzyme. Both activities also co-migrated during isoelectric focusing, and had similar, but not identical, heat inactivation profiles. The poly(C) polymerase reaction was identical with the normal CMP-incorporating activity of tRNA nucleotidyltransferase with regard to pH and temperature optima, requirements for a divalent cation and an RNA acceptor, and nonlinear double reciprocal plots with respect to varying CTP concentrations. However, the poly(C) polymerase activity also differed in several properties, including its response to divalent cations, ATP, salt, and organic solvents. Normal CMP incorporation into tRNA-C was most active with Mg2+, was stimulated by ATP and salt, and was inhibited by a variety of organic solvents. In contrast, the poly(C) polymerase reaction was most active with Mn2+, was inhibited by ATP and salt, and was stimulated by organic solvents. The poly(C) polymerase activity utilized intact tRNA-C-C-A, tRNA-C-C, 5 S RNA, and rRNA as acceptors for CMP incorporation. In the presence of Mg2+ the rate of the anomalous reaction was generally only a few per cent of CMP incorporation into the presumed normal acceptor, tRNA-C, although 5 S RNA was 15% as active. In all cases, the poly(C) polymerase reaction was stimulated 2- to 3-fold by 1 mm Mn2+ and the normal reaction was inhibited, so that under these conditions the best substrate was 5 S RNA. Extended nucleotide incorporation into tRNA-C-C-A or rRNA was found only in the presence of CTP, whereas incorporation of AMP and UMP was limited or nonexistent. These data indicate that liver tRNA nucleotidyltransferase, like several other enzymes which synthesize phosphodiester bonds, can also act as a homopolymer polymerase. The relation of this activity to other known poly(C) polymerases and its possible mechanism of action are also discussed.