Nucleic Acids Research, 2003, Vol. 31, No. 4 1197-1207
© 2003 Oxford University Press
Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli
Department of Biological Science, Shimane University, Matsue, 690-8504, Japan and 1 Institut für Biochemie, Universität Würzburg, Biozentrum, Am Hubland, D-97074 Würzburg, Germany
*To whom correspondence should be addressed. Tel: +81 852 32 6431; Fax: +81 852 32 6449; Email: akama{at}life.shimane-u.ac.jp
The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNASer(CGA), an Arabidopsis intron-containing tRNATyr(GTA) and an Arabidopsis intron-containing tRNAMet(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of ß-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNASer gene and the GUS reporter gene resulted in 
10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNATyr or tRNAMet genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNAMet(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNATyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNATyr and tRNAMet were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.
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