Identification of Two Escherichia coli Pseudouridine Synthases That Show Multisite Specificity for 23S RNA

Abstract

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