Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli.
tRNA pseudouridine 55 (psi 55) synthase, the enzyme that is specific for the conversion of U55 to psi 55 in the m5U psi CG loop in most tRNAs, has been purified from Escherichia coli and cloned. On SDS gels, a single polypeptide chain with a mass of 39.7 kDa was found. The gene is a previously described open reading frame, p35, located at 68.86 min on the E. coli chromosome between the infB and rpsO genes. The proposed name for this gene is truB. There is very little protein sequence homology between the truB gene product and the hisT (truA) product, which forms psi in the anticodon arm of tRNAs. However, there was high homology with a fragment of a Bacillus subtilis gene that may produce the analogous enzyme in that species. The cloned gene was fused to a 5'-leader coding for a (His)6 tract, and the protein was overexpressed > 400-fold in E. coli. The recombinant protein was purified to homogeneity in one step from a crude cell extract by affinity chromatography using a Ni(2+)-containing matrix. The SDS mass of the recombinant protein was 41.5 kDa, whereas that calculated from the gene was 37.3. The recombinant protein was specific for U55 in tRNA transcripts and reacted neither at other sites for psi in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments. The enzyme did not require either a renatured RNA structure or Mg2+, and prior formation of m5U was not required. Stoichiometric formation of psi occurred with no requirement for an external source of energy, indicating that psi synthesis is thermodynamically favored.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006639:Histidine; D019753:Intramolecular Lyases / Q000235:genetics* / Q000302:isolation & purification; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D011560:Pseudouridine / Q000096:biosynthesis*; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D010455:Peptides; D012360:RNA, Transfer, Phe; D012366:RNA, Transfer, Val; D011994:Recombinant Proteins; D011560:Pseudouridine; C033223:polyhistidine; D006639:Histidine; D012343:RNA, Transfer; D007535:Isomerases; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D019753:Intramolecular Lyases