Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes

• Published in: Nucleic acids research Vol. 11; no. 3; pp. 707 - 718
• Main Authors: Fournier, M, Haumont, E, de Henau, S, Gangloff, J, Grosjean, H
• Format: Journal Article
• Language: English
• Published: England 11.02.1983
• Subjects: Animals; Anticodon - genetics; Base Sequence; Endonucleases; Female; Kinetics; Microinjections; Nucleic Acid Conformation; Oocytes - metabolism; Ovum - metabolism; Ribonuclease T1; RNA Processing, Post-Transcriptional; RNA, Transfer - genetics; RNA, Transfer, Amino Acyl - genetics; Saccharomyces cerevisiae - genetics; Single-Strand Specific DNA and RNA Endonucleases; Xenopus
• ISSN: 0305-1048

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected 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 tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (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 adenosine and cytosine were not.