Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli.
The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA(Ser)(CGA), an Arabidopsis intron-containing tRNA(Tyr)(GTA) and an Arabidopsis intron-containing tRNA(Met)(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 beta-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 tRNA(Ser) gene and the GUS reporter gene resulted in approximately 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 tRNA(Tyr) or tRNA(Met) genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA(Met)(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 tRNA(Tyr) 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 tRNA(Tyr) and tRNA(Met) 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.
Akama
Kazuhito
K
Department of Biological Science, Shimane University, Matsue, 690-8504, Japan. akama@life.shimane-u.ac.jp
Beier
Hildburg
H
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D018389:Codon, Nonsense / Q000235:genetics*; D046508:Culture Techniques; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D018546:Hypocotyl / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D030821:Plants, Genetically Modified; D014176:Protein Biosynthesis / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000235:genetics; D012362:RNA, Transfer, Ser / Q000737:chemistry / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic
D018389:Codon, Nonsense; D012322:RNA Precursors; D012358:RNA, Transfer, Met; D012362:RNA, Transfer, Ser; D012365:RNA, Transfer, Tyr; D011993:Recombinant Fusion Proteins; D012343:RNA, Transfer; D005966:Glucuronidase