In order to study how Escherichia coli leucyl-tRNA synthetase recognizes tRNALeu and discriminates it from the other two class II tRNAs, tRNASer and tRNATyr, various mutations were introduced into class II tRNA transcripts. The discriminator base A73, but not the anticodon sequence, was found to serve as a critical recognition element of tRNALeu. A base substitution at the invariant nucleotide A14, but not at any of the other nucleotides characteristic of the E. coli tRNALeu isoaeceptors among the three class II tRNAs, caused significantly damaged aminoacylation with leucine. A two base pair deletion in the long variable arm also resulted in no significant decrease of activity. Transplanting the three tertiary elements characteristic of E. coli tRNALeu (i.e. the location of the G18G19 sequence in the D-loop, the A15·U48 base-pair and the stem pairing pattern of the long variable arm) besides the discriminator base change introduced the leucine charging activity in terms of Vmax/Km, up to 0.1 of that for the normal sequence of tRNALeu into both tRNASer and tRNATyr. These results indicate that A73 and A14 (or its vicinity) are involved in recognition by leucyl-tRNA synthetase, and that several tertiary elements play a significant role in the discrimination of tRNALeu from the other two class II tRNAs. © 1993 Academic Press, Inc.
