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

Abstract

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.

Figure 1

Cloverleaf structures of suppressor tRNAs derived from E.coli tRNA^Gln. The mutated anticodon sequences and the C9 to A9 mutation are circled.

Figure 2

Schematic representation of the luciferase reporter mRNA encoding a Renilla luciferase–firefly luciferase (RLucFLuc) fusion protein. Internal stop codon mutations in the firefly luciferase gene are indicated. The luciferase reporter mRNA has two termination signals at the 3′-terminus separated by a UUC codon (…UAAUUCUAG…polyA…; termination codons are underlined).

Figure 3

Acid urea PAGE/northern blot analysis of hsup2am, hsup2oc and hsup2op tRNAs. Total tRNA was isolated under acidic conditions and separated by acid urea PAGE. Suppressor tRNAs were visualized by RNA blot hybridization using a 5′-³²P-labeled oligonucleotide complementary to nucleotides 57–72 of E.coli tRNA^Gln. A 5′-³²P-labeled oligonucleotide complementary to nucleotides 7–22 of the human tRNA^Ser was used as internal standard for quantitation of RNA and aminoacylation levels by PhosphorImager analysis.

Figure 4

Amber, ochre and opal suppression in HEK293T cells. Immunoblot analysis of proteins isolated from cells co-transfected with plasmids carrying the genes encoding the luciferase reporter, hsup2/C32A38am, hsup2/C32A38oc or the hsup2/C32A38op tRNAs and, when present, E.coli GlnRS. The RLucFLuc fusion protein was detected with an anti-FLuc antibody and E.coli GlnRS was detected with an anti-His4-antibody. An antibody against β-actin was used as a loading control. RLucFLuc, full-length fusion protein; RLucFLuc^*, truncated RLucFLuc fusion protein.

Figure 5

Acid urea PAGE/northern blot analysis of additional mutants derived from hsup2am, hsup2oc and hsup2op tRNAs. (A) amber suppressor series; (B) ochre suppressor series; (C) opal suppressor series. Suppressor tRNAs were visualized by RNA blot hybridization using a 5′-³²P-labeled oligonucleotide complementary to nucleotides 57–72 of tRNA^Gln. A 5′-³²P-labeled oligonucleotide complementary to nucleotides 7–22 of the human tRNA^Ser was used as internal standard (data not shown) for quantitation of RNA and aminoacylation levels by PhosphorImager analysis.

Figure 6

β-galactosidase activity in cell extracts of E.coli with an amber mutation in the chromosomal β-galactosidase gene transformed with plasmids carrying the hsup2am, hsup2/C32A38am, hsup2oc and hsup2/C32A38oc tRNA genes. Values represent the averages of at least three independent experiments.