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 tRNA at the 3''-terminal nucleoside. The first involved the incubation of an enzymatically abbreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from E. 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-COH 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 tRNA in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNA 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 tRNA terminating in 2''- and 3''-O-methyladenosine produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNA 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 tRNA-C-COH. Although incubation of tRNA-C-COH with 3 aminoacylated nucleoside 5''-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNA 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.