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

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 tRNAPheGAA or tRNACysGCA 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 a synthetic promoter, derived from the promoter sequence of the E. coli lipoprotein gene. The tRNAPhe suppressor (tRNAPheCUA) is 54-100% efficient in vivo, while the tRNACys suppressor (tRNACysCUA) 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 tRNAPheCUA and tRNACysCUA insert phenylalanine and cysteine, respectively. To demonstrate the potential of these suppressors, tRNAPheCUA and tRNACysCUA have been used to effect amino acid substitutions at specific sites in the E. coli lac repressor.