Mutations of Arabidopsis thaliana pre-tRNA(Tyr) affecting pseudouridylation of U35.

The structural and sequence requirements for the biosynthesis of tRNA(Tyr) pseudouridine (psi 35) have been studied. Nucleotide substitution at the 32nd position slightly reduced modification efficiency in the case of transition (C32 to U32) while transversion (C32 to G32) had no effect on the modification process in wheat germ extract. Insertion of one nucleotide into the anticodon stem caused a 2-fold reduction of modification efficiency. Mutants with a partially deleted 12 nt long intron of pre-tRNA(Tyr) exhibited different effects: deletion of 5 nt (7 nt long intron) gave only a reduction in pseudouridylation while deletion of 7 nt (5 nt long intron) almost completely abolished the reaction. The generated mini-substrate consisting of pre-tRNA(Tyr) anticodon stem and intron sequence was partially modified which proved that the crucial elements for recognition of psi 35 introduction had to present in this construct.