Reactions at the 3′ Terminus of Transfer Ribonucleic Acid

The extents of AMP, CMP, and UMP incorporation catalyzed by tRNA nucleotidyltransferase were examined under a variety of experimental conditions. At low levels of enzyme nucleotide incorporation into tRNApCpC, tRNApC, and tRNApX in the presence of both ATP and CTP led to the formation of the normal product, tRNApCpCpA. However, when ATP or UTP were present without CTP, tRNApCpA, tRNApCpU, or tRNApU were also synthesized. At high concentrations of tRNA nucleotidyltransferase, a wide variety of unusual tRNA molecules were made. These included molecules with AMP, CMP, or UMP in each, and sometimes all, of the terminal positions, as well as tRNA chains containing as many as 8 CMP residues. These altered tRNA molecules should prove valuable for elucidating the biological function of the -pCpCpA terminus of tRNA. Evidence is also presented that apparently homogeneous preparations of tRNA nucleotidyltransferase contain a poly(C) polymerase activity which utilized as substrates intact tRNA, rRNA, and tRNAs without complete termini. With rRNA as substrate, molecules containing as many as 40 CMP residues could be made. AMP and UMP were generally not added to rRNA, but in the presence of CTP, AMP residues could be incorporated. The properties of this poly(C) polymerase activity were similar to those of the CMP-incorporating activity of tRNA nucleotidyltransferase and differed from those of mammalian poly(C) polymerase suggesting that contamination of the enzyme preparation was probably not involved. A rapid procedure for the location of nucleotide residues added to RNA chains is also described.