CONSTRUCTION OF ESCHERICHIA-COLI AMBER SUPPRESSOR TRANSFER-RNA GENES .2. SYNTHESIS OF ADDITIONAL TRANSFER-RNA GENES AND IMPROVEMENT OF SUPPRESSOR EFFICIENCY

Using synthetic oligonucleotides, we have constructed 17 tRNA suppressor genes from Escherichia coli representing 13 species of tRNA. We have measured the levels of in vivo suppression resulting from introducing each tRNA gene into E. coli via a plasmid vector. The suppressors function at varying efficiencies. Some synthetic suppressors fail to yield detectable levels of suppression, whereas others insert amino acids with greater than 70% efficiency. Results reported in the accompanying paper demonstrate that some of these suppressors insert the original cognate amino acid, whereas others do not. We have altered some of the synthetic tRNA genes in order to improve the suppressor efficiency of the resulting tRNAs. Both tRNACUAHis and tRNACUAGlu were altered by single base changes, which generated -A-A- following the anticodon, resulting in a markedly improved efficiency of suppression. The tRNACUAPro was inactive, but a hybrid suppressor tRNA consisting of the tRNACUAPhe anticodon stem and loop together with the remainder of the tRNAPro proved highly efficient at suppressing nonsense codons. Protein chemistry results reported in the accompanying paper show that the altered tRNACUAHis and the hybrid tRNACUAPro insert only histidine and proline, respectively, whereas the altered tRNACUAGlu inserts principally glutamic acid but some glutamine. Also, a strain deficient in release factor 1 was employed to increase the efficiency of weak nonsense suppressors. © 1990 Academic Press Limited.