PURIFICATION, CLONING, AND PROPERTIES OF THE TRANSFER-RNA PSI-55 SYNTHASE FROM ESCHERICHIA-COLI

tRNA pseudouridine 55 (Psi 55) synthase, the enzyme that is specific f or the conversion of U55 to Psi 55 in the m(5)U Psi CG loop in most tR NAs, 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 m in on the E. coli chromosome between the infB and rpsO genes. The prop osed name for this gene is truB. There is very little protein sequence homology between the truB gene product and the hisT (truA) product, w hich 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 overexpresse d >400-fold in E. coli. The recombinant protein was purified to homoge neity in one step from a crude cell extract by affinity chromatography using a Ni2+-containing matrix. The SDS mass of the recombinant prote in was 41.5 kDa, whereas that calculated from the gene was 37.3. The r ecombinant protein was specific for U55 in tRNA transcripts and reacte d neither at other sites for Psi in such transcripts nor with transcri pts of 16S or 23S ribosomal RNA or subfragments. The enzyme did not re quire either a renatured RNA structure or Mg2+, and prior formation of m(5)U was not required. Stoichiometric formation of Psi occurred with no requirement for an external source of energy, indicating that Psi synthesis is thermodynamically favored.