A CELL-FREE YELLOW LUPIN EXTRACT CONTAINING ACTIVITIES OF PSEUDOURIDINE-35 AND PSEUDOURIDINE-55 SYNTHASES

Plant cytoplasmic tyrosine tRNA was pseudouridylated at three differen t positions: 35, 39 and 55. These pseudouridines were introduced by th ree different enzymes pseudouridine synthases. Variants of the Arabido psis thaliana pre-tRNA(Tyr) were constructed that allow to monitor spe cifically pseudouridylation at different nucleotide positions. Using s uch RNAs to assay pseudouridine synthesis we have prepared an extract from Lupinus luteus cv. Ventus seeds containing activities of at least Psi 35 and Psi 55 synthases. This is the first report describing the preparation of the lupin seed extract that specifically modifies plant pre-tRNA(Tyr) transcribed by T7 RNA polymerase. U35 is converted to P si 35 only in an intron-dependent manner, while pseudouridylation of U 55 is insensitive to the presence or absence of an intron.