Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and...

In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo⁵U) or 5-methoxycarbonylmethoxyuridine (mcmo⁵U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho⁵U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho⁵U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho⁵U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho⁵C synthesis in E. coli Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo⁵U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho⁵U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo⁵U as that observed for mo⁵U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho⁵U synthesis in E. coli to the same extent as yegQ Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho⁵U biosynthesis in bacteria is similar to that of ho⁵C, but additional genes and substrates are required for complete modification.IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.