title: Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.
abstract: The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan takahito.mukai@riken.jp.
authors.forename: Takahito
authors.initials: T
authors.lastname: Mukai
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ohtake
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Mihoko
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hayashi
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Fumie
authors.initials: F
authors.lastname: Iraha
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kira
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yanagisawa
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hoshi
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan kensaku.sakamoto@riken.jp.
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry; D005798:Genes, Bacterial; D020043:Genes, Essential; D006709:Homoarginine / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering; D020543:Proteome / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D013489:Suppression, Genetic
chemical_list: D000596:Amino Acids; D003062:Codon; D029968:Escherichia coli Proteins; C090663:N(6)-(1-iminoethyl)lysine; D020543:Proteome; D006709:Homoarginine; D012343:RNA, Transfer; D001120:Arginine; D000604:Amino Acyl-tRNA Synthetases; D008239:Lysine