Reassigning cysteine in the genetic code of Escherichia coli.
We investigated directed deviations from the universal genetic code. Mutant tRNAs that incorporate cysteine at positions corresponding to the isoleucine AUU, AUC, and AUA and methionine AUG codons were introduced in Escherichia coli K12. Missense mutations at the cysteine catalytic site of thymidylate synthase were systematically crossed with synthetic suppressor tRNACys genes coexpressed from compatible plasmids. Strains harboring complementary codon/anticodon associations could be stably propagated as thymidine prototrophs. A plasmid-encoded tRNACys reading the codon AUA persisted for more than 500 generations in a strain requiring its suppressor activity for thymidylate biosynthesis, but was eliminated from a strain not requiring it. Cysteine miscoding at the codon AUA was also enforced in the active site of amidase, an enzyme found in Helicobacter pylori and not present in wild-type E. coli. Propagating the amidase missense mutation in E. coli with an aliphatic amide as nitrogen source required the overproduction of Cys-tRNA synthetase together with the complementary suppressor tRNACys. The toxicity of cysteine miscoding was low in all our strains. The small size and amphiphilic character of this amino acid may render it acceptable as a replacement at most protein positions and thus apt to overcome the steric and polar constraints that limit evolution of the genetic code.
Döring
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Groupe de Chimie Biologique, Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, 75015 Paris, France.
Marlière
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P
D000581:Amidohydrolases / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics; D003062:Codon / Q000235:genetics; D003433:Crosses, Genetic; D003545:Cysteine / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005815:Genetic Code / Q000235:genetics*; D016480:Helicobacter pylori / Q000201:enzymology; D007532:Isoleucine / Q000235:genetics; D008715:Methionine / Q000235:genetics; D008957:Models, Genetic; D020125:Mutation, Missense; D012350:RNA, Transfer, Cys / Q000235:genetics; D013489:Suppression, Genetic; D013940:Thymidylate Synthase / Q000235:genetics
D003062:Codon; D012350:RNA, Transfer, Cys; D007532:Isoleucine; D008715:Methionine; D013940:Thymidylate Synthase; D000581:Amidohydrolases; C019665:amidase; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase; D003545:Cysteine