Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells.
We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.
Köhrer
Caroline
C
Department of Biology, Room 68-671, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Sullivan
Eric L
EL
RajBhandary
Uttam L
UL
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D018388:Codon, Terminator / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017930:Genes, Reporter; D016153:Genes, Suppressor; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013489:Suppression, Genetic
D018388:Codon, Terminator; D012343:RNA, Transfer; D008156:Luciferases; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase