Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA

Several putative Escherichia coli pseudouridine (Psi) synthases have been identified by iterative searching of genomic databases for ORFs homologous to known Psi synthases [Gustafsson et al. (1996) Nucleic Acids Res. 24, 3756-3762]. Of these, yceC and yfil were proposed to encode Psi synthases which modify 23S rRNA. In the present work, yceC and yfil were cloned and overexpressed in E. coli, and the encoded enzymes, YceC and Yfil, were purified to homogeneity. Both proteins converted Urd residues of rRNA to Psi, thus confirming their identities as Psi synthases. However, in in vitro experiments both enzymes extensively modified Urd residues of both 23S rRNA and 16S rRNA. Gene-disruption of yceC resulted in the absence of Psi modification at positions U955, 2504,and 2580 of 23S RNA, thus identifying these sites as in vivo targets for YceC. Likewise, yfil disruption resulted in the absence of Psi modification at positions U1911, 1917, and possibly 1915 of 23S RNA. Disruption of yceC did not affect the growth under the conditions tested, whereas yfil-disrupted cells showed a dramatic decrease in growth rate. Since YceC and Yfil hypermodify RNA in vitro, factors in addition to ribonucleotide sequence must contribute to the in vivo specificity of these enzymes.