Discrimination between D- and L-tyrosyl transfer ribonucleic acids in peptide chain elongation

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Abstract
D.sbd.Tyr.sbd.tRNA can take part in peptide bond formation with N-AcPhe-tRNA on Escherichia coli ribosomes programmed with the hexanucleotide UUUUAC. None of the steps leading to peptide bond formation exhibit high stereoselectivity. Ternary complex formation with EF-Tu[elongation factor Tu].cntdot.GTP favors L.sbd.Tyr.sbd.tRNA by a factor > 25. The complex formed with D.sbd.Tyr.sbd.tRNA was not protected from hydrolysis, which suggests that the D-amino acid is improperly bound to the protein. The rate of EF.sbd.Tu.sbd.promoted dipeptide formation was 30-fold faster with L.sbd.Tyr.sbd.tRNA. The ratio of moles of GTP hydrolyzed to dipeptide formed was 1.4 for L.sbd.Tyr.sbd.tRNA and 4 for D.sbd.Tyr.sbd.tRNA. The excess of GTP hydrolyzed to peptide bonds formed is evidence for kinetic proofreading in AA(aminoacyl)-tRNA selection. The combined effects of the partial discrimination at each stage, from the aminoacylation to the peptide formation, favor L-tyrosine by a factor > 104 and would virtually exclude D-tyrosine from being incorporated under conditions in which L-tyrosine was also present.