Nonsense suppression in archaea.
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.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Arpita
A
Bhattacharya
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Caroline
C
Köhrer
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Debabrata
D
Mandal
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 bhandary@mit.edu.
Uttam L
UL
RajBhandary
D001105:Archaea / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018389:Codon, Nonsense; D018388:Codon, Terminator; D004926:Escherichia coli / Q000378:metabolism; D016153:Genes, Suppressor; D019622:Haloferax volcanii / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009675:Novobiocin / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012343:RNA, Transfer / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013489:Suppression, Genetic; D013936:Thymidine / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014498:Uracil / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism
D018389:Codon, Nonsense; D018388:Codon, Terminator; D009675:Novobiocin; D012694:Serine; D014498:Uracil; D014364:Tryptophan; D012343:RNA, Transfer; D001616:beta-Galactosidase; D013936:Thymidine
Haloferax volcanii; haloarchaea; inducible tRNA expression; nonsense suppression; suppressor tRNA