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

Abstract

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.

Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity.

“…Our methodology is analogous to the one developed recently by Bruce Annealing Uhlenbeck (1982); which takes advantage of the properties of T4 RNA ligase and T4 polynucleotide kinase. For yeast tRNAAsP, the experimental procedures were as described in detail by Carbon et al (1982); for yeast tRNAArg and yeast tRNALeu we followed the procedure described by Fournier et al (1983). Figure 3 illustrates the last step of such a procedure with several anticodon-modified yeast tRNAAsP Post-transcriptional modifications of chimerical yeast tRNA after microinjection into frog oocytes Kinetics of queuine insertion in yeast tRNAAsP.…”

“…To study this, we have taken advantage of the possibility of modifying the anticodon sequence of a tRNA in vitro (Carbon et al, 1982 Bruce andUhlenbeck, 1982;Fournier et al, 1983). We have created a series of chimeric yeast tRNAAsP (normal anticodon GUC) by varying the immediate vicinity of G34.…”

Recognition of bases in Escherichia coli tRNA(Gln) by glutaminyl-tRNA synthetase: a complete identity set.

“…technology (reviewed in Cedergren and Grosjean, 1987) which has previously been applied to the formation of tRNAs with altered anticodon (Kaufmann and Littauer, 1974;Bruce and Uhlenbeck, 1982a,b;Fournier et al, 1983;Ohtsuka et al, 1983;Schulman and Pelka, 1983; Bare and Uhlenbeck, 1985) or T loop sequences (Carbon and Ebel, 1987). The approach involves controlled nucleolytic cleavage of tRNA followed by ligation to specific oligoribonucleotides and resealing of the tRNA with T4 RNA ligase.…”

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys

“…Here we describe the application of the RNA mutagenic technology to the construction of a potential UGA suppressor with anticodon UCA, by base substitution in yeast tRNACyS. This methodology, dependent largely on the properties of RNA ligase and polynucleotide kinase from phage T4 [5, 61, was initially developed by the group of Uhlenbeck, and applied successfully to the modification of several yeast tRNAs: tRNAPhe [4], tRNAASp [8] and tRNAkg [9].Essentially three reasons led us to select yeast tRNACy" as an interesting species for modification. Among the anticodon changes that may be easily introduced, one should lead to a UGA suppressor species, the activity of which may be readily assayed in vivo as well as in vitro, The normal tRNACYs anticodon, GCA, is conveniently split by ribonuclease TI.…”

“…Here we describe the application of the RNA mutagenic technology to the construction of a potential UGA suppressor with anticodon UCA, by base substitution in yeast tRNACyS. This methodology, dependent largely on the properties of RNA ligase and polynucleotide kinase from phage T4 [5, 61, was initially developed by the group of Uhlenbeck, and applied successfully to the modification of several yeast tRNAs: tRNAPhe [4], tRNAASp [8] and tRNAkg [9].…”