The discrimination mechanism between tRNASer and tRNATyr was studied using various in vitro transcripts of E. coli tRNATyr variants. The insertion of only two nucleotldes into the variable stem of tRNATyr generates serine charging activity. The acceptor activities of some of the tRNATyr mutants with insertions in the long variable arm were enhanced by changes in nucleotides at positions 9 and/or 20B, which are possible elements for dictating the orientation of the long variable arm. These findings suggest that the long variable arm is involved in recognition by seryl-tRNA synthetase in spite of sequence and length variations shown within tRNASer isoacceptors, and eventually serves as a determinant for selection from other tRNA species. Changing the anticodon from GUA to the serine anticodon GGA resulted in a marked decrease in tyrosine charging activity, but this mutant did not show any serine charging activity. The discriminator base, the fourth base from the 3′ end of tRNA, was also Important for aminoacylation with tyrosine. Complete specificity change in vitro was facilitated by insertion of three nucleotides into the variable arm plus two nucleotide changes at positions 9 and 73.
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