CONSTRUCTION OF ESCHERICHIA-COLI AMBER SUPPRESSOR TRANSFER-RNA GENES .3. DETERMINATION OF TRANSFER-RNA SPECIFICITY

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNACUAAla2, tRNACUAGly1, tRNACUAHisA, tRNACUALys and tRNACUAProH, inserted the predicted amino acid. The class II suppressors, tRNACUAGluA, tRNACUAGly2 and tRNACUAIle1 were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNACUAArg, tRNACUAAspM, tRNACUAIle2, tRNACUAThr2, tRNACUAMet(m) and tRNACUAVal inserted predominantly lysine. © 1990 Academic Press Limited.