Nucleic Acids Research, Vol 25, Issue 22 4493-4499, Copyright © 1997 by Oxford University Press
HF Becker, Y Motorin, RJ Planta and H Grosjean
The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w
and CBF5) display a remarkable sequence homology with Escherichia coli
tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene YNL292w
coding for one of these proteins was cloned in an E.coli expression vector
downstream of a His6-tag. The resulting recombinant protein (Pus4) was
expressed at high level and purified to homogeneity by metal affinity
chromatography on Ni2+-NTA-agarose, followed by ion-exchange chromatography
on MonoQ. The purified Pus4p catalyzes the formation of pseudouridine-55 in
T7 in vitro transcripts of several yeast tRNA genes. In contrast to the
known yeast pseudouridine synthase (Pus1) of broad specificity, no other
uridines in tRNA molecules are modified by the cloned recombinant
tRNA:Psi55 synthase. The disruption of the corresponding gene YNL292w in
yeast, which has no significant effect on the growth of yeast cells, leads
to the complete disappearance of the Psi55 formation activity in a cell-
free extract. These results allow the formal identification of the protein
encoded by the yeast ORF YNL292w as the only enzyme responsible for the
formation of Psi55 which is almost universally conserved in tRNAs. The
substrate specificity of the purified YNL292w-encoded recombinant protein
was shown to be similar to that of the native protein present in yeast cell
extract. 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 Psi55 formation in
tRNAs of both cellular compartments.
ARTICLES
The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs
Laboratoire d'Enzymologie et Biochimie Structurales du CNRS, Avenue de la Terrasse, Batiment 34, F-91198 Gif-sur-Yvette, France.
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