Construction of two Escherichia coli amber suppressor genes: tRNAPheCUA and tRNACysCUA.

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Abstract
Amber suppressor genes corresponding to Escherichia coli tRNAPhe and tRNACys have been constructed for use in amino acid substitution studies as well as protein engineering. The genes for either .**GRAPHIC**. or .**GRAPHIC**. both with the anticodon 5'' CTA 3'' were assembled from four to six oligonucleotides, which were annealed and ligated into a vector. the suppressor genes are expressed constitutively from synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor .**GRAPHIC**. is 54-100% efficient in vivo, while the tRNACys suppressor .**GRAPHIC**. is 17-50% efficient. To verify that the suppressors insert the predicted amino acids, both genes were used to suppress an amber mutation in a protein coding sequence. NH2-terminal sequence analysis of the resultant proteins revealed that .**GRAPHIC**. and .**GRAPHIC**. insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, .**GRAPHIC**. and .**GRAPHIC**. have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.