Nonsense suppression in archaea

Significance Three stop codons are used as translation termination signals within the mRNA. Nonsense suppression is defined as the read through of stop codons in an mRNA by a class of mutant tRNAs called nonsense suppressor tRNAs. Although much is known about nonsense suppression in bacteria (e.g., Escherichia coli ) and eukaryotes (e.g., yeast) and although suppressor tRNAs have served as invaluable tools in bacterial and bacterial viral genetics, very little is known about nonsense suppression in archaea, the third kingdom of life. We describe here for the first time, to our knowledge, the suppression of all three stop codons in Haloferax volcanii , a halophilic archaeon, by suppressor tRNAs derived from serine or tyrosine tRNA. 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 archaeon Haloferax volcanii are 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 in H. volcanii . Additionally, transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in the pyrE2 gene 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.