Functional redundancy in tRNA dihydrouridylation

Nucleic Acids Res. 2024 Jun 10;52(10):5880-5894. doi: 10.1093/nar/gkae325.


Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.

MeSH terms

  • Bacillus subtilis* / enzymology
  • Bacillus subtilis* / genetics
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Gene Expression
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Transfer* / genetics
  • RNA, Transfer* / metabolism
  • Uridine* / analogs & derivatives
  • Uridine* / metabolism


  • Bacterial Proteins
  • RNA, Bacterial
  • RNA, Transfer
  • Uridine