Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the β-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeonHaloferax volcaniiare active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons inH. volcanii. Additionally, transformation of aΔpyrE2 H. volcaniistrain with plasmids carrying the genes for apyrE2amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in thepyrE2gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.
589: Lowering the hurdle for nonsense suppressor tRNA delivery through sequence optimization
