Reactions at the 3′ Terminus of Transfer Ribonucleic Acid

The specificity and properties of anomalous AMP incorporation catalyzed by rabbit liver tRNA nucleotidyltransferase were studied. A single AMP residue could be added to the 3' terminus of a variety of RNA molecules, including rRNA and 5 S RNA. In addition, tRNA molecules containing 1 or 3 terminal CMP residues, tRNA-C and tRNA-C-C-C, were also active as acceptors. The rate of AMP incorporation into these anomalous substrates was considerably more rapid if the RNA acceptor contained a terminal CMP residue. Thus, the rate of AMP incorporation into tRNA-C-C was about 20-fold more rapid than into tRNA-C-A and tRNA-C-U. Nevertheless, at high levels of enzyme it was possible to convert almost completely the latter molecules to tRNA-C-A-A and tRNA-C-U-A. All the anomalous reactions were stimulated by the presence of Mn2+, as much as 10-fold in the case of 5 S RNA, whereas normal AMP incorporation was inhibited. Under optimal conditions, wheat germ 5 S RNA was as active a substrate as tRNA-C-C. Furthermore, the two RNA acceptors had similar apparent Km values. These results are discussed in terms of the recognition site on the RNA acceptors and the importance of the 3'-terminal residue. Evidence is also presented that the 3'-terminal moiety of wheat germ 5 S RNA is predominantly CMP. In addition, an improved method for determining the location of nucleotide residues incorporated into RNA is described.