A DUAL-SPECIFICITY PSEUDOURIDINE SYNTHASE - AN ESCHERICHIA-COLI SYNTHASE PURIFIED AND CLONED ON THE BASIS OF ITS SPECIFICITY FOR PSI-746 IN23S RNA IS ALSO SPECIFIC FOR PSI-32 IN TRNA(PHE)

An Escherichia coli pseudouridine (Psi) synthase, which forms both Psi 746 in E. coli 23S ribosomal RNA and Psi 32 in tRNA(Phe), has been is olated and cloned. The enzyme contains 219 amino acids and has a calcu lated MW of 24,432 Da. Amino acid sequence comparison with the three o ther Psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the c atalysis of a common reaction. The gene was cloned behind a (His)(6) l eader for affinity purification. Upon overexpression, most of the enzy me remained soluble in the cell cytoplasm and could be purified to hom ogeneity on a Ni2+-containing resin. The enzyme reacted with both full -length 23S RNA or a fragment from residues 1-847, forming 1 mol Psi/m ol RNA at position 746, a normal site for Psi. The enzyme has no depen dence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg 2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were no t modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, wer e modified with a yield of 1 mol Psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the Psi to be at positi on 32, a normal site for Psi in this tRNA. Both 23S rRNA Psi 746 and t RNA Psi 32 occur in single-stranded segments of the same sequence, Psi UGAAAA, closed by a stem. Therefore, this synthase may require for re cognition only a short stretch of primary sequence 3' to the site of p seudouridylation. This is the first example of a dual-specificity modi fying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The e ssentiality of these Psi residues can now be assessed by disruption of the synthase gene.