Wobble Uridine C5 Modification in Escherichia coli tRNA

Abstract

The post-transcriptional modification of the wobble uridine in E. coli tRNAs is significant for facilitating non-Watson-Crick base pairing which modulates decoding capabilities. One of the common wobble uridine modification, uridine 5-oxyacetic acid (or also called as 5-carboxymethoxyuridine, cmo5U) biogenesis via 5-hydroxyuridine (ho5U) formation is widely conserved in gram negative bacteria. tRNA bound enzyme structure determination by X-ray crystal structures of wobble uridine hydroxylases YceA (O2 dependent), YegQ (prephenate dependent), and carboxymethyltransferase CmoB from E. coli have been attempted to elucidate the unknown chemical mechanism. The in vitro activity of YceA to conduct reaction on stem loop tRNAAla(UGC), tRNAPro(UGG), and tRNAThr(UGU) was confirmed by LC-MS analysis. Similarly, CmoB coupled assay was carried out with YceA which generated cmo5U in full length and stem loop tRNA. Overall, the YceA and CmoB activity has been reconstituted. The requirements for both enzymes’ substrate specificity was noted while details remain elusive, including the reaction mechanism of YceA.