Modification of the anticodon triplet of E.coli tRNAMetf by replacement with trimers complementary to non-sense codons UAG and UAA.
E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.
D003062:Codon; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis*; D012358:RNA, Transfer, Met; D012259:Ribonuclease, Pancreatic
D003062:Codon; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D004722:Endoribonucleases; D012259:Ribonuclease, Pancreatic