An unnatural base pair for incorporating amino acid analogs into proteins.
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.
Hirao
Ichiro
I
Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. ihirao@postman.riken.go.jp
Ohtsuki
Takashi
T
Fujiwara
Tsuyoshi
T
Mitsui
Tsuneo
T
Yokogawa
Tomoko
T
Okuni
Taeko
T
Nakayama
Hiroshi
H
Takio
Koji
K
Yabuki
Takashi
T
Kigawa
Takanori
T
Kodama
Koichiro
K
Yokogawa
Takashi
T
Nishikawa
Kazuya
K
Yokoyama
Shigeyuki
S
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
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