Identity of tRNA for yeast tyrosyl-tRNA synthetase: Tyrosylation is more sensitive to identity nucleotides than to structural features

The specific aminoacylation of tRNA by yeast tyrosyl-tRNA synthetase does n ot rely on the presence of modified residues in tRNA(Tyr), although such re sidues stabilize its structure. Thus, the major tyrosine identity determina nts were searched by the in vitro approach using unmodified transcripts pro duced by T7 RNA polymerase. On the basis of the tyrosylation efficiency of tRNA variants, the strongest determinants are base pair C1-G72 and discrimi nator residue A73 (the 5'-phosphoryl group on C1, however, is unimportant f or tyrosylation). The three anticodon bases G34, U35, and A36 contribute al so to the tyrosine identity, but to a lesser extent, with 034 having the mo st pronounced effect. Mutation of the GUA tyrosine anticodon into a CAU met hionine anticodon, however, leads to a loss of tyrosylation efficiency simi lar to that obtained after mutation of the C1-G72 or A73 determinants. Tran splantation of the six determinants into four different tRNA frameworks and activity assays on heterologous Escherichia coli and Methanococcus jannasc hii tRNA(Tyr) confirmed the completeness of the tyrosine set and the eukary otic character of the C1-G72 base pair. On the other hand, it was found tha t tyrosine identity in yeast does not rely on fine architectural features o f the tRNA, in particular the size and sequence of the D-loop. Noticeable, yeast TyrRS efficiently charges a variant of E. coli tRNA(Tyr) With a large extra-region provided its G1-C72 base pair is changed to a C1-G72 base pai r. Finally, tyrosylation activity is compatible with a +1 shift of the anti codon in the 3'-direction but is strongly inhibited if this shift occurs in the opposite 5'-direction.