CHARACTERIZATION OF YEAST PROTEIN DEG1 AS PSEUDOURIDINE SYNTHASE (PUS3) CATALYZING THE FORMATION OF PSI(38) AND PSI(39) IN TRANSFER-RNA ANTICODON LOOP

The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli, The results show that the DEG1-disru pted yeast strain lacks synthase activity for the formation of pseudou ridines Psi(38),,,, and Psi(39) in tRNA whereas the other activities, specific for Psi formation at positions 13, 27, 28, 32, 34, 35, 36, an d 55 in tRNA, remain unaffected, Also, the His(6)-tagged recombinant y east Deg1p expressed in E., coli as well as a protein fusion with prot ein A in yeast display the enzymatic activity only toward Psi(38) and Psi(39) formation in different tRNA substrates, Therefore, Deg1p is th e third tRNA:pseudouridine synthase (Pus3p) characterized so far in ye ast, Disruption of the DEG1 gene is not lethal but reduces considerabl y the yeast growth rate, especially at an elevated temperature (37 deg rees C), Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the pseudour idine residues present (or absent) in selected naturally occurring cyt oplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of Psi(38),, and Psi(39)-synthesizing activity in bot h of these two cellular compartments, The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied, The results indicate th e existence of subtle differences in the tRNA recognition by yeast Pus 3p and by its homologous tRNA:pseudouridine synthase truA from E., col i (initially called hisT or PSU-I gene product).