Five-base codons for incorporation of nonnatural amino acids into proteins.
Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon-anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA(UACCG) chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN(1)N(2), where N(1) and N(2) indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.
Hohsaka
T
T
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Ashizuka
Y
Y
Murakami
H
H
Sisido
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D000926:Anticodon / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003062:Codon / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000235:genetics; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics
D000596:Amino Acids; D000926:Anticodon; D001426:Bacterial Proteins; D003062:Codon; D012333:RNA, Messenger; D012365:RNA, Transfer, Tyr; D014443:Tyrosine; D019809:Streptavidin