Studies on aminoacyl tRNA synthetases and transfer RNA in living Xenopus laevis oocytes

Abstract

The microinjection of transfer RNA into amphibian oocytes permits one to study under in vivo conditions the reactions that affect this important macromolecule. A comparative study has been carried out between the in vivo and in vitro specificity of the aminoacylation reacton. The results obtained show that modifications of the tRNA structure affect aminoacyl-tRNA synthetase recognition in the same fashion in both conditions. The in vivo aminoacylation was not affected by the presence of puromycin (0.5mM) or cycloheximide (0.1 mM) which completely inhibited oocyte protein synthesis. An interesting difference was obtained between the in vivo and in vitro aminoacylation of tRNA with regards to temperature requirements. While the in vivo reaction was optimal at 25 degrees and was totally inhibited at 37 degrees, the in vitro was optimal, at the latter temperature. The inhibition of the in vivo reaction at 37 degrees was not due to inactivation of the enzyme. The transfer of the amino acid moiety to nascent proteins was studied by measuring the transfer of radioactivity from injected (14C) phenylalanyl-tRNA into hot trichloroacetic acid precipitable material. It was found that 30% or more of the amino acid became incorporated into oocyte proteins and that this incorporation was due to direct transfer from the aminoacyl-tRNA and was inhibited by puromycin and cycloheximide.