| Abstract
| - The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does not rely on thepresence of modified residues in tRNATyr, although such residues stabilize its structure. Thus, the majortyrosine identity determinants were searched by the in vitro approach using unmodified transcripts producedby T7 RNA polymerase. On the basis of the tyrosylation efficiency of tRNA variants, the strongestdeterminants are base pair C1−G72 and discriminator residue A73 (the 5‘-phosphoryl group on C1,however, is unimportant for tyrosylation). The three anticodon bases G34, U35, and A36 contribute alsoto the tyrosine identity, but to a lesser extent, with G34 having the most pronounced effect. Mutation ofthe GUA tyrosine anticodon into a CAU methionine anticodon, however, leads to a loss of tyrosylationefficiency similar to that obtained after mutation of the C1−G72 or A73 determinants. Transplantation ofthe six determinants into four different tRNA frameworks and activity assays on heterologous Escherichiacoli and Methanococcus jannaschii tRNATyr confirmed the completeness of the tyrosine set and theeukaryotic character of the C1−G72 base pair. On the other hand, it was found that tyrosine identity inyeast does not rely on fine architectural features of the tRNA, in particular the size and sequence of theD-loop. Noticeable, yeast TyrRS efficiently charges a variant of E. coli tRNATyr with a large extra-regionprovided its G1−C72 base pair is changed to a C1−G72 base pair. Finally, tyrosylation activity iscompatible with a +1 shift of the anticodon in the 3‘-direction but is strongly inhibited if this shift occursin the opposite 5‘-direction.
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