Transfer RNA-mediated suppression of amber stop codons in transgenic Arabidopsis thaliana

An artificial amber suppressor tRNA^Leu gene (supL.) was physically linked to a mutated gus reporter gene, p35S-gus(amL), which was inactivated by an amber stop codon (amL). Upon introduction into Arabidopsis thaliana, the presence of the supL. gene was found to be correlated with cytotoxic effects observed during tissue culture and in mature plants. Those primary transformants that displayed cytotoxic symptoms were shown by X-Gluc staining to express GUS as a result of amber stop codon suppression in vivo. Phenotypically normal lines were found by RT-PCR to express supL. GUS activity above background level was barely detectable in these plants, indicating a low level expression of supL. However, the remaining suppressor activity was still sufficient to transactivate an amber-mutated male sterility gene, pA9-barnase(amL1) when combined within the same plant by crossing. The suppressor tRNA^Leu gene may thus be used in transgenic plants for gene transactivation.     

Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem

Threonyl-tRNA synthetase, a class II synthetase, uses a unique zinc ion to discriminate against the isosteric valine at the activation step. The crystal structure of the enzyme with an analog of seryl adenylate shows that the noncognate serine cannot be fully discriminated at that step. We show that hydrolysis of the incorrectly formed ser-tRNA(Thr) is performed at a specific site in the N-terminal domain of the enzyme. The present study suggests that both classes of synthetases use effectively the ability of the CCA end of tRNA to switch between a hairpin and a helical conformation for aminoacylation and editing. As a consequence, the editing mechanism of both classes of synthetases can be described as mirror images, as already seen for tRNA binding and amino acid activation.     

RNA-mediated genetic transformation in Aspergillus nidulans

We developed a model-system for correcting genetic alterations of an Aspergillus nidulans strain (ribo, paba, bio, w, Acr) by treating protoplasts with total RNA extracted from another A. nidulans strain bearing wild type alleles for the same genetic markers. The results revealed the occurrence of a true genetic transformation. The phenomenon was RNA-dependent since it was abolished by pancreatic ribonuclease treatment. The term retrotransformation is proposed since the RNA messages artificially inserted into the mutant protoplast cells restore the wild type chromosomal information.