Biogenesis and iron-dependency of ribosomal RNA hydroxylation

Abstract

Post-transcriptional modifications of ribosomal RNAs (rRNAs) are involved in ribosome biogenesis and fine-tuning of translation. 5-Hydroxycytidine (ho5C), a modification of unknown biogenesis and function, is present at position 2501 of Escherichia coli 23S rRNA. We conducted a genome-wide screen in E. coli to identify genes required for ho5C2501 formation, and found a previously-uncharacterized gene, ydcP (renamed rlhA), iron-sulfur cluster (isc) genes, and a series of genes responsible for prephenate biosynthesis, indicating that iron-sulfur clusters and prephenate are required for ho5C2501 formation. RlhA interacted with precursors of the 50S ribosomal subunit, suggesting that this protein is directly involved in formation of ho5C2501. RlhA belongs to a family of enzymes with an uncharacterized peptidase U32 motif and conserved Cys residues in the C-terminal region. These elements were essential for ho5C2501 formation. We also found that the frequency of ho5C2501 is modulated by environmental iron concentration. Together, our results reveal a novel biosynthetic pathway for RNA hydroxylation and its response to iron.

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

“…coli versus 18 copies in B. subtilis) (1). As recently described by Kimura et al (21) and shown in Fig. 2, E. coli contains four peptidase U32 genes as follows: rlhA (ydcP), yegQ, and a putative operon containing the two adjacent genes yhbU and yhbV.…”

“…2B, and also weak homology to Fe-S clustercontaining proteins in both bacteria and archaea. In addition, Kimura et al (21) published that the peptidase U32 gene rlhA was required for ho 5 C synthesis in E. coli rRNA. Thus, it was likely that one or both of these genes were required for ho 5 U synthesis in tRNA in B. subtilis.…”

“…E. coli iscS mutant synthesizes cmo 5 U but at a reduced level. An important finding from Kimura et al (21) was that ho 5 C synthesis in rRNA in E. coli is significantly dependent on Fe-S cluster biogenesis, specifically for genes in the isc operon. This is consistent with the earlier mentioned PSI-BLAST results.…”

“…One of the most intriguing aspects of the cmo 5 U modification in tRNA was the observation that chorismic acid or a downstream metabolite not only was required for the synthesis of the methylene carbon of cmo 5 U but also was necessary for wt levels of ho 5 U (19). Kimura et al (21) have recently provided strong evidence that prephenic acid is the metabolite required for ho 5 C synthesis in rRNA; thus, it is reasonable to suspect it is also utilized by YegQ in the synthesis of ho 5 U. HPLC analysis of an aroD E. coli mutant from the Keio collection showed the expected complete absence of cmo 5 U and the presence of ho 5 U at reduced levels ( Fig. 5A), confirming the original results from Hagervall et al in S. Typhimurium (19).…”

“…While genes have been identified for the biosynthesis of the alkylation steps in the ho 5 U-derived modification (xo 5 U) pathways, those involved in the hydroxylation have remained unknown. Kimura et al recently reported that the hydroxylation step in the synthesis of structurally similar 5-hydroxycytidine (ho 5 C) in rRNA is dependent on the rlhA gene (formerly ydcP) (21). RlhA is a member of a group of proteins found in bacteria annotated as U32 peptidases (NCBI conserved domain accession number COG0826) but with no experimentally known function.…”