Functional assay of tRNA molecules transcribed from a purified gene

Purified tRNA genes are expressed when microinjected into the nucleus of X.laevis oocytes. In this paper we describe a method to assay the capacity to be aminoacylated of the tRNA transcribed in the frog oocytes. The method exploits the radiochemical purity of the transcript and relies on the binding of aminoacyl-tRNA but not of uncharged tRNA to purified elongation factor EF-TU. We also present some preliminary results on several single point mutants of tRNA^Pro from Caenorhabditis elegans. We show that nucleotide 73 of tRNA^Pro can be substituted by any other nucleotide without loss of acceptor activity. A doublemutant, causing transition from G₄₅G₄₆ to A₄₅₄₆ has lost acceptor activity. Also inactive is a mutant carrying an insertion of a single base in the anticodon loop.