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. 2011 Dec 6;108(49):19593-8.
doi: 10.1073/pnas.1112352108. Epub 2011 Nov 28.

Molecular basis of dihydrouridine formation on tRNA

Futao Yu  1 , Yoshikazu Tanaka, Keitaro Yamashita, Takeo Suzuki, Akiyoshi Nakamura, Nagisa Hirano, Tsutomu Suzuki, Min Yao, Isao Tanaka
Affiliations

Molecular basis of dihydrouridine formation on tRNA

Futao Yu et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overall structure of TthDus-tRNA complex (A) Ribbon diagram. The region of Ala171-Ile180, which was disordered in the RNA-free TthDus, is shown in red. FMN is shown as sticks. (B) Electrostatic surface potential of TthDus. Positively and negatively charged surfaces are colored blue and red, respectively (formula image). (C) Overall interaction scheme among TthDus, FMN, and tRNA. Hydrogen bonds, π-stacking interaction, and covalent bond between TthDus and tRNA are shown as black, green, and red arrows, respectively.
Fig. 2.
Fig. 2.
T- and D-loop recognition mechanisms (A) Conformational differences between the bound Tth-tRNAPhe and yeast tRNAPhe. The cartoon model is colored according to the rmsd between two tRNA molecules from blue (0.59 Å) to red (6.49 Å). All atoms in the ribose and phosphate moieties were used for calculation. TthDus is shown as a white surface. (B) Superposition of the T-arms and D-loops of Tth-tRNAPhe (red) and yeast tRNAPhe (blue). The numbers represent the tRNA base positions. (C) Recognition of the elbow region. Polar interactions between TthDus residues and tRNA nucleotides (red dashed line) and those within tRNA nucleotides (yellow dashed line) are shown as stereo views. The cartoon models of TthDus and tRNA are also shown in orange and green, respectively.
Fig. 3.
Fig. 3.
Structure of TthDus-tRNA fragment complex. (A) Close-up view of the U20 binding site (stereo views). Electron density (Fo-Fc map) of the unknown cofactor molecule is also shown as green mesh (contoured at 3σ). Dotted lines represent hydrogen bonds. Water molecule is shown as a red ball. (B) Covalent bond between Cys93 and U20. The 2Fo-Fc map is also shown (contoured at 2σ).
Fig. 4.
Fig. 4.
Mutation analyses (A) Evaluation of the covalent complex formation of TthDus mutants by SDS-PAGE. Results for the mutants in the active site are shown. The results for the Y103A mutant, which cannot form a covalent complex, are also shown. Other mutants are shown in Fig. S5. (B) Native PAGE of the TthDus mutants incubated with tRNAArg extracted from E. coli dus-deficient strain. The results for wild type with transcribed tRNAArg are also shown. Lane 1: 20 pmol tRNA, lane 2; 20 pmol tRNA + 20 pmol TthDus mutant, lane 3; 20 pmol tRNA + 40 pmol TthDus mutant, lane 4; 20 pmol tRNA + 60 pmol TthDus mutant. (C) Enzymatic activity of each EcYjbN mutant. The amounts of dihydrouridine introduced into E. coli tRNAArg2 and tRNAArg3 are shown relative to the wild type.
Fig. 5.
Fig. 5.
Proposed reaction mechanism of Dus (A) Distances between Cys93, target U20, and FMN in the structure of TthDus-tRNA fragment complex. (B) Schematic representation of proposed mechanism for uridine reduction of Dus. Hydride attacks C6 of uridine with a slight positive charge (left). The generated electron pair at C5 attacks the distal hydrogen of Cys93 (center). Dihydrouridine is generated (right).
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