Construction, expression, and function of a new yeast amber suppressor, tRNATrpA

The number of different tRNA species in Saccharomyces cerevisiae known to be capable of suppressing termination of translation at UAG, UAA, and UGA codons is limited to those which insert tyrosine, leucine, and serine. Suppressor tRNAs that insert other amino acids, even those whose anticodons differ from the expected recognition sequences for nonsense codons by a single nucleotide, have never been identified via classical genetic analysis. We have used site-directed mutagenesis to convert the anticodon of a cloned tRNATrp gene from CCA to CTA with the expectation that this gene would produce tRNA molecules capable of interacting with the UAG terminator codon. We show that this form of the gene can be transcribed and spliced in vitro to produce mature tRNA with the expected base sequence. The putative suppressor gene has been introduced into several S. cerevisiae host strains using the centromere vector YCp19. Efficient suppression of amber mutations met8-1, tyr7-1, and lys2-801 results from the presence of the CTA form of tDNATrp. Two UAA mutants, leu2-1 and ade2-101, and the UGA marker his4-260 are not suppressed.