In vitro conversion of a methionine to a glutamine-acceptor tRNA.
A derivative of Escherichia coli tRNAfMet containing an altered anticodon sequence, CUA, has been enzymatically synthesized in vitro. The variant tRNA was prepared by excision of the normal anticodon, CAU, in a limited digestion of intact tRNAfMet with RNase A, followed by insertion of the CUA sequence into the anticodon loop with T4 RNA ligase and polynucleotide kinase. The altered methionine tRNA showed a large enhancement in the rate of aminoacylation by glutaminyl-tRNA synthetase and a large decrease in the rate of aminoacylation by methionyl-tRNA synthetase. Measurement of kinetic parameters for the charging reaction by the cognate and noncognate enzymes revealed that the modified tRNA is a better acceptor for glutamine than for methionine. The rate of mischarging is similar to that previously reported for a tryptophan amber suppressor tRNA containing the anticodon CUA, su+7 tRNATrp, which is aminoacylated with glutamine both in vivo and in vitro [Yaniv, M., Folk, W. R., Berg, P., & Soll, L. (1974) J. Mol. Biol. 86, 245-260; Yarus, M., Knowlton, R. E., & Soll, L. (1977) in Nucleic Acid-Protein Recognition (Vogel, H., Ed.) pp 391-408, Academic Press, New York]. The present results provide additional evidence that the specificity of aminoacylation by glutaminyl-tRNA synthetase is sensitive to small changes in the nucleotide sequence of noncognate tRNAs and that uridine in the middle position of the anticodon is involved in the recognition of tRNA substrates by this enzyme.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000926:Anticodon; D001483:Base Sequence; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000235:genetics*; D005973:Glutamine / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis; D010761:Phosphorus Radioisotopes; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis / Q000378:metabolism*; D012358:RNA, Transfer, Met
D000926:Anticodon; D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D005973:Glutamine; D000255:Adenosine Triphosphate