THE YEAST GENE YNL292W ENCODES A PSEUDOURIDINE SYNTHASE (PUS4) CATALYZING THE FORMATION OF PSI(55) IN BOTH MITOCHONDRIAL AND CYTOPLASMIC TRANSFER-RNAS

The protein products of two yeast Saccharomyces cerevisiae genes (YNL2 92w and CBF5) display a remarkable sequence homology with Escherichia coil tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene Y NL292w coding for one of these proteins was cloned in an E. coli expre ssion vector downstream of a His(6)-tag. The resulting recombinant pro tein (Pus4) was expressed at high level and purified to homogeneity by metal affinity chromatography on Ni2+-NTA-agarose, followed by ion-ex change chromatography on MonoQ. The purified Pus4p catalyzes the forma tion of pseudouridine-55 in T-7 in vitro transcripts of several yeast tRNA genes. In contrast to the known yeast pseudouridine synthase (Pus 1) of broad specificity, no other uridines in tRNA molecules are modif ied by the cloned recombinant tRNA:psi(55) synthase. The disruption of the corresponding gene YNL292w in yeast, which has no significant eff ect on the growth of yeast cells, leads to the complete disappearance of the psi(55) formation activity in a cell-free extract. These result s allow the formal identification of the protein encoded by the yeast ORF YNL292w as the only enzyme responsible for the formation of psi(55 ) which is almost universally conserved in tRNAs. The substrate specif icity of the purified YNL292w-encoded recombinant protein was shown to be similar to that of the native protein present in yeast cell extrac t. Chemical mapping of pseudouridine residues in both cytoplasmic and mitochondrial tRNAs from the yeast strain carrying the disrupted gene reveals that the same gene product is responsible for psi(55) formatio n in tRNAs of both cellular compartments.