Post-transcriptlonal modification of the wobble nucletide in anticodon-substituted yeast tRNAIIArgafter microinjection intoxenopus laevis oocytes

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Abstract
An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAλJg by NCG tnnucleotide (N=A, C, G or U) is described. Partial digestion with Si-nuclease and Ti-RNAase provides fragments which, when annealed together, form an “anticodon-deprived” yeast tRNA4?g. A novel anticodon, phosphorylated with (λ2P) label on its 51 terminal residue, is then inserted using T4-RNA ligase. Such “anticodon-substituted” yeast tRNAj* are micromjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34***) in yeast tRNAλJg it is efficiently modified into mosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5u. In contrast, when a cytosme (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosme and cytosme were not.