title: An unnatural base pair for incorporating amino acid analogs into proteins.
abstract: An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.
authors.affiliation: Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. ihirao@postman.riken.go.jp
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohtsuki
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Mitsui
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yokogawa
authors.forename: Taeko
authors.initials: T
authors.lastname: Okuni
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Koji
authors.initials: K
authors.lastname: Takio
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kodama
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokogawa
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001483:Base Sequence; D002474:Cell-Free System; D003062:Codon; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D014644:Genetic Variation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D014764:Viral Proteins
chemical_list: D000596:Amino Acids; D003062:Codon; D012333:RNA, Messenger; D014764:Viral Proteins; D014443:Tyrosine; D004247:DNA; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C087259:3-chlorotyrosine