Enzymatic replacementin vitroof the first anticodon base of yeast tRNA^Asp: application to the study of tRNA maturationin vivo, after microinjection into frog oocytes

Abstract

A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.

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

“…Jean VACHER, Henri GROSJEAN, Suzanne DE HENAU, Jacqueline FINELLI, and Richard H. BUCKINGHAM Institut de Biologie Physico-Chimique, Paris; and Laboratoire de Chimie Biologique, Rhode St-Genese (Received July 8,1983) -EJB 83 0724…”

“…1) were isolated by partial digestion of tRNACyS. Preliminary experiments with different enzyme concentrations and times of incubation were performed to determine optimum conditions for digestion: 4 units Tl/mg tRNA, incubated for 90min at 4 "C; lo4 units S1 (Miles)/mg tRNA, incubated for 20 min at 37 "C. Other conditions were as described previously [8] except that S1 digestions included 10% glycerol. The TI fragment OH-C,,-A,,-OH and the S1 fragment pG1 -U,,-OH were purified on a polyacrylamide slab gel (13.5 % w/v acrylamide, 0.68 % bisacrylamide in 100 mM Tris/borate buffer, pH 8.3, 2.5 mM Na,EDTA and 8.3 M urea).…”

“…c) Modification of the base in the 'wobble' position. Posttranscriptional modification of tRNACy" (anticodon UCA) and tRNACyS (anticodon GCA) in the first anticodon position was investigated as previously described for tRNAAsp [8]. The protocol described above was used (Fig.…”

“…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].…”