Preparation of Escherichia coli tRNAs Terminating in Modified Nucleosides by the Use of CTP(ATP): TRNA Nucleotidyltransferase and Polynucleotide Phosphorylase

Two procedures were investigated for the modification of tRNAs at the 3′-terminal nucleoside. The first involved the incubation of an enzymatically abbreviated tRNA (IRNA-C-C_OH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2′- or 3′-deoxyadenosine 5′-triphosphate as substrates, but affected incorporation of the 2′- and 3′-O-methyladenosine triphosphates onto tRNA-C-C_OH to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP):tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-C_OH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2′- and 3′-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2′- and 3′-O-methyladenosine produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2′- and 3′-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomeric aminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto IRNA-C-C_OH. Although incubation of tRNA-C-C_OH with three aminoacylated nucleoside 5′-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2′-deoxy-3′-O-l-phenylalanyladenosine 5′-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.