Transfer RNA-mediated suppression of stop codons in protoplasts and transgenic plants

Abstract

We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNA(Leu) gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, UUA and UCA) that recognize, respectively, the amber, ochre and opal stop codons. The suppression activity of these genes was detected by their ability to restore transient beta-glucuronidase (GUS) expression in tobacco protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA gene and a GUS gene containing a premature amber stop codon showed up to 20-25% of the activity found in protoplasts transfected with the functional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1%. This system could be adapted to examine transcription, processing or aminoacylation of tRNAs in plant cells. In addition, phenotypically normal, fertile tobacco plants expressing a stably incorporated amber suppressor tRNA gene have been obtained. This suppressor tRNA can be used to transactivate a target gene containing a premature amber stop codon by a factor of at least several hundred-fold.

Enrichment of cereal protein lysine content by altered tRNAlys coding during protein synthesis

“…Protein synthesis naturally incurs errors, at frequencies for particular codons approaching 1% (Kurland et al, 1997;Parker, 1992). As has been extensively studied in prokaryotes and to a limited extent in yeasts and animal cells, the expression of altered tRNAs in plant cells and plants heightens a natural lack of fidelity of protein synthesis (Betzner et al, 1997;Carneiro et al, 1993;Chen Z. et al, 1998;Choisne et al, 1997;Franklin et al, 1992;Ulmasov and Folk, 1995 Furthermore, as the changes in the anticodons of these tRN-A lys species reduce the efficiency of tRNA amino-acylation by the plant lysyl tRNA synthetase (Folk et al, unpublished data), it might be possible to selectively enhance the amino-acylation of these tRNAs and to target their utilization during protein synthesis by expressing high levels of free lysine and /or the lysyl tRNA synthetase in endosperm. As one of the problems associated with expressing high levels of free lysine in plant seeds is the production of lysine catabolites from the excess lysine (Galili, 2002;Mazur et al, 1999) enhancing lysine incorporation into proteins might provide a shunt and alleviate this problem.…”

The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

“…Tobacco leaf protoplast preparation, protoplast electroporation, total protein extracts, and GUS activity measurements were done as previously described (Carneiro et al, 1993). Purification of tobacco protoplast mitochondria was principally done as described above for Arabidopsis mitochondria, except that extraction was done in 60 mL of extraction buffer and the Percoll gradients were spun in 2-mL tubes containing 450 pL Of 5O%, 700 pL of 25%, and 500 pL of 14% Percoll.…”

Plant viruses as model systems for the study of non-canonical translation mechanisms in higher plants

“…One additional functional aspect is its capacity to act as a shifty stop codon and promote ribosomal frameshifting, as in the expression of the PVM CP-12K trans-frame protein (Gramstat et al, 1993). Further insight into the in vivo activity and specificity of UAA-, UAG-or UGAdecoding plant tRNAs will be gained by the expression of appropriate gene fusions with reporter genes by transient expression in protoplasts or after stable transformation and regeneration of transgenic plants (Tacke et al, 1990;Priifer, 1992;Carneiro et al, 1993).…”