Molecular basis of dihydrouridine formation on tRNA

Futao Yu, Yoshikazu Tanaka, Keitaro Yamashita, Takeo Suzuki, Akiyoshi Nakamura, Nagisa Hirano, Tsutomu Suzuki, Min Yao, Isao Tanaka

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

Abstract

Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recognizes the elbow region composed of the kissing loop interaction between T- and D-loops in tRNA, pulling U20 into the catalytic center for reduction. Although distortion of the D-loop structure was observed upon binding of Dus to tRNA, the canonical D-loop/T-loop interaction was maintained. These results were consistent with the observation that Dus preferentially recognizes modified rather than unmodified tRNAs, indicating that Dus introduces D20 by monitoring the complete L-shaped structure of tRNAs. In the active site, U20 is stacked on the isoalloxazine ring of FMN, and C5 of the U20 uracil ring is covalently cross linked to the thiol group of Cys93, implying a catalytic mechanism of D20 formation. In addition, the involvement of a cofactor molecule in uracil ring recognition was proposed. Based on a series of mutation analyses, we propose a molecular basis of tRNA recognition and D formation catalyzed by Dus.

Original languageEnglish
Pages (from-to)19593-19598
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number49
DOIs
Publication statusPublished - 2011 Dec 6
Externally publishedYes

Keywords

  • Protein-tRNA complex
  • RNA modification
  • Substrate recognition
  • X-ray crystallography

ASJC Scopus subject areas

  • General

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