The chapter describes the preparation of isomeric tRNAs terminating in 2'- and 3'-deoxyadenosine. The modification procedure is illustrated both for unfractionated Escherichia coli tRNA and for E. coli tRNA Trp. The principle states that by limited digestion of tRNAs with purified venom exonuclease, it is possible to effect partial removal of the cytidine and adenosine moieties from the 3' terminus without significant hydrolysis of nucleotides from the adjacent double-stranded portion of the accepter stem. While hydrolysis does not proceed uniformly over the entire population of treated tRNAs, especially where unfractionated material is utilized, the average extent of hydrolysis can be monitored conveniently. Reconstitution to afford tRNA-C-COH is subsequently achieved by incubation of the nuclease-treated samples with CTP (but not ATP) in the presence of CTP(ATP):tRNA nucleotidyltransferase. Further incubation in the presence of 2'- or 3'-deoxyadenosine 5'-triphosphate affords the modified tRNAs of interest, purification of which can be effected chromatographically on DEAE-cellulose and DBAE-cellulose columns.
ASJC Scopus subject areas
- Molecular Biology