Amber suppressor tRNAs of phage T5: Gene constructs and suppression efficiency in vivo

The genes of 23 phage T5 amber suppressor tRNAs were constructed using site -directed mutagenesis and were cloned in plasmid pGFIB1 under control of th e lipoprotein gene constitutive promoter. Suppression efficiency was measur ed in vivo after introducing recombinant plasmids into E. coli strains bear ing mutations in the lacI gene. Strong amber suppressors (not less than 40% efficiency of the wild type) were obtained with tRNAs specific for amino a cids Ala, Gin, Gly, His, Lys, Ser, and Tyr. Suppressor activity was conside rably enhanced by additional substitutions which restored pairing in the D stem (tRNA(CUA)(Asp)) and in the T stem (tRNA(CUA)(Glu)) outside the antico don, as well as by substitution U32C in the anticodon loop of tRNA(CUA)(Arg ).