Construction of a Systematic Set of tRNA Mutants by Ligation of Synthetic Oligonucleotides into Defined Single-Stranded Gaps

Abstract

A series of mutant tRNA genes has been constructed by site-directed mutagenesis in pOP203, a colE1 derivative carrying a transcription unit under control of the lacUV5 promoter. These mutant genes include all possible amber suppressing variants of tRNA^Trp with single nucleotide substitutions at anticodon loop positions 32, 37, and 38 (numbered from the 5′end), and all possible paired base substitutions in the three base pairs nearest the anticodon loop. G at position 38 was not recovered as a single mutation, but rather in conjunction with an undirected mutation to T at position 32. The singly mutated G38 tRNA may not be active, though all the other tRNA derivatives are functional in the translation of amber codons. To construct the mutants, we ligated a synthetic deoxyoligonucleotide into a precisely formed single-stranded gap covering the anticodon arm region DNA, in an otherwise double-stranded fragment containing the tRNA^Trp gene. The resulting heteroduplex was then ligated into the plasmid and introduced into Escherichia coli. This method of mutagenesis is simple, reproducible, and highly tolerant of varying degrees of heteroduplex in the gap, variations in temperature of ligation, and changes in the oligonucleotide concentration. Mutagenesis does not require a 5′-phosphorylated oligonucleotide. These qualities suit the gap method for intensive study of a region by site-directed mutagenesis.