PURIFICATION, CLONING, AND PROPERTIES OF THE 16S-RNA PSEUDOURIDINE-516 SYNTHASE FROM ESCHERICHIA-COLI

Pseudouridine (Psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes, In Escherichia coil smal l subunit RNA, there is only one Psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this sin gle Psi residue, the enzyme catalyzing its formation was purified and cloned, The enzyme contains 231 amino acids and has a calculated molec ular mass of 25 836 Da. It converts U516 in E. coil 16S RNA transcript s into Psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 3 0S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of Psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as w as RNA(1-526) and the RNP particle made from it. 23S RNA and tRNA(Val) transcripts were also inactive. These results suggest that Psi format ion in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same dir ection as, the bicyclomycin resistance gene. The gene was cloned behin d a (His)(6) leader for affinity purification. Virtually all of the ov erexpressed protein was found in inclusion bodies but could be purifie d to homogeneity on a Ni2+ containing resin. Over 200 mg of pure prote in could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and Psi sequence analysis established that B. sub tilis has the equivalent of Psi-516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enz yme and the two tRNA Psi synthases which have been cloned up to now.