A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya.
Putative pseudouridine synthase genes are members of a class consisting of four subgroups that possess characteristic amino acid sequence motifs. These genes have been found in all organisms sequenced to date. In Escherichia coli, 10 such genes have been identified, and the 10 synthase gene products have been shown to function in making all of the pseudouridines found in tRNA and ribosomal RNA except for tRNA(Glu) pseudouridine13. In this work, a protein able to make this pseudouridine was purified by standard biochemical procedures. Amino-terminal sequencing of the isolated protein identified the synthase as YgbO. Deletion of the ygbO gene caused the loss of tRNA(Glu) pseudouridine13 and plasmid-borne restoration of the structural gene restored pseudouridine13. Reaction of the overexpressed gene product, renamed TruD, with a tRNA(Glu) transcript made in vitro also yielded only pseudouridine13. A search of the database detected 58 homologs of TruD spanning all three phylogenetic domains, including ancient organisms. Thus, we have identified a new wide-spread class of pseudouridine synthase with no sequence homology to the previously known four subgroups. The only completely conserved sequence motif in all 59 organisms that contained aspartate was GXKD, in motif II. This aspartate was essential for in vitro activity.
Kaya
Yusuf
Y
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Ofengand
James
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001224:Aspartic Acid / Q000502:physiology; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000502:physiology; D005057:Eukaryotic Cells / Q000201:enzymology*; D017353:Gene Deletion; D005798:Genes, Bacterial; D016678:Genome; D006836:Hydro-Lyases / Q000737:chemistry / Q000145:classification* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D011560:Pseudouridine / Q000032:analysis / Q000096:biosynthesis; D012352:RNA, Transfer, Glu / Q000096:biosynthesis / Q000737:chemistry; D017423:Sequence Analysis, RNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012352:RNA, Transfer, Glu; D011560:Pseudouridine; D001224:Aspartic Acid; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase