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

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 (moU) or 5-methoxycarbonylmethoxyuridine (mcmoU). 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 (hoU) remain unknown. Here, a number of genes in and are identified that are required for wild-type (wt) levels of hoU. The operon is identified in as important for the biosynthesis of hoU. Both and are homologs to peptidase U32 family genes, which includes the gene required for hoC synthesis in Deletion of either or , or both, gives a 50% reduction in moU tRNA levels. In , was found to be the only one of four peptidase U32 genes involved in hoU synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmoU as that observed for moU in the mutants. By analyzing the genomic context of homologs, the ferredoxin YfhL is shown to be required for hoU synthesis in to the same extent as Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that hoU biosynthesis in bacteria is similar to that of hoC, but additional genes and substrates are required for complete modification. 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 and 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.