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, 47 (20), 10942-10955

Structure of tRNA Methyltransferase Complex of Trm7 and Trm734 Reveals a Novel Binding Interface for tRNA Recognition

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Structure of tRNA Methyltransferase Complex of Trm7 and Trm734 Reveals a Novel Binding Interface for tRNA Recognition

Akira Hirata et al. Nucleic Acids Res.

Abstract

The complex between Trm7 and Trm734 (Trm7-Trm734) from Saccharomyces cerevisiae catalyzes 2'-O-methylation at position 34 in tRNA. We report biochemical and structural studies of the Trm7-Trm734 complex. Purified recombinant Trm7-Trm734 preferentially methylates tRNAPhe transcript variants possessing two of three factors (Cm32, m1G37 and pyrimidine34). Therefore, tRNAPhe, tRNATrp and tRNALeu are specifically methylated by Trm7-Trm734. We have solved the crystal structures of the apo and S-adenosyl-L-methionine bound forms of Trm7-Trm734. Small angle X-ray scattering reveals that Trm7-Trm734 exists as a hetero-dimer in solution. Trm7 possesses a Rossmann-fold catalytic domain, while Trm734 consists of three WD40 β-propeller domains (termed BPA, BPB and BPC). BPA and BPC form a unique V-shaped cleft, which docks to Trm7. The C-terminal region of Trm7 is required for binding to Trm734. The D-arm of substrate tRNA is required for methylation by Trm7-Trm734. If the D-arm in tRNAPhe is docked onto the positively charged area of BPB in Trm734, the anticodon-loop is located near the catalytic pocket of Trm7. This model suggests that Trm734 is required for correct positioning of tRNA for methylation. Additionally, a point-mutation in Trm7, which is observed in FTSJ1 (human Trm7 ortholog) of nosyndromic X-linked intellectual disability patients, decreases the methylation activity.

Figures

Figure 1.
Figure 1.
Characterization of the recombinant Trm7–Trm734 (A) SDS-PAGE (12.5%) of purified recombinant Trm7–Trm734. The gel was stained with Coomassie Brilliant Blue. (B) Cloverleaf structure of Saccharomyces cerevisiae tRNAPhe transcript. Three SAM-dependent tRNA methyltransferases, Trm7–Trm732, Trm7–Trm734 and TrmD, catalyze formation of Cm32, Gm34 and m1G37, respectively. (C) Time-dependent methyl transfer activity of Trm7–Trm734 using S. cerevisiae tRNAPhe transcript (YF, green triangles), YF with Cm32 (red squares), YF with m1G37 (cyan circles) and YF with both Cm32 and m1G37 (orange cross marks). Error-bars indicate the standard deviation calculated from the results of three independent experiments. (D) Methyl-group acceptance activities of mutant tRNAPhe transcripts were measured. G34 in the wild-type transcript was replaced by A, U or C. Left, the transcripts were analyzed by 10% PAGE (7 M urea). The gel was stained with methylene blue. Right, autoradiogram of the same gel.
Figure 2.
Figure 2.
Structure of Trm7–Trm734. (A) Ribbon diagram of the X-ray structure of Trm7–Trm734. Trm7 is colored red. The three domains of Trm734 (BPA, BPB and BPC) and linker regions are colored green, blue, yellow and black, respectively. The structures on the right are rotated through −90° along the horizontal axis. (B) Schematic representation of Trm7 and Trm734 with domain boundaries. The dotted lines show the regions of Trm7 which are invisible. (C) Overlay of the envelope shape of Trm7–Trm734 calculated with DAMMIN using the SAXS data and the CORAL model of Trm7–Trm734 based on its X-ray structure.
Figure 3.
Figure 3.
Structures of Trm7 and Trm734 subunits in the Trm7–Trm734 complex. (A) Ribbon diagram of the Trm7 structure. (B) Ribbon diagram of Trm734. Three WD40 domains—BPA, BPB and BPC—are colored, green, blue and yellow, respectively. The linker regions are colored black. Close-up view enclosed in a dotted square indicates one WD40 blade formed by four antiparallel β strands.
Figure 4.
Figure 4.
SAM bound form of Trm7–Trm734. (A) Ribbon and surface schematic of the overall structure of Trm7–Trm734 in complex with SAM. Trm7, and BPA, BPB and BPC of Trm734 are colored red, and green, blue, and yellow, respectively. SAM is depicted as a stick model. (B) SAM binding pocket in Trm7. Close-up view of SAM binding pocket is shown in a dotted square. The amino acid residues responsible for the interactions with SAM are highlighted as stick models (green).
Figure 5.
Figure 5.
The V-shaped cleft of Trm734 docks to Trm7. (A) Docking of Trm7 onto Trm734 through the V-shaped cleft. The V-shaped cleft is opened to 85° (dotted line). (B) Close-up view of the ribbon diagram of the interaction of Trm7 (red) with BPA (green) and BPC (yellow) of Trm734. The stick models (dark-gray) show the C-terminal region (N233-A256) of Trm7 essential for the interaction between Trm7 and Trm734. The structure of Escherichia coli RlmE (cyan) is superimposed onto that of Trm7 (red). (C) 12% SDS-PAGE of purified Δ260–310 Trm7 mutant. The gel was stained with Coomassie Brilliant Blue. (D) About 12% SDS-PAGE of purified Δ233–310 Trm7 mutant. (E) Relative methyl-transfer activities of the wild-type Trm7–Trm734 (WT) and deletion mutants (Δ233–310 Trm7 and Δ260–310 Trm7). The initial velocity of the WT for tRNAPhe transcript is expressed as 100.0%.
Figure 6.
Figure 6.
Search for the RNA-binding mode of Trm7–Trm734. (A) Surface model of Trm7–Trm734 colored according to the electrostatic potential represented as a gradient from negative (red) to positive (blue). Saccharomyces cerevisiae tRNAPhe is placed along the distribution of positively charged surface of Trm7–Trm734–SAM. A line diagram of S. cerevisiae tRNAPhe is colored orange. The D-arm of S. cerevisiae tRNAPhe is colored black. (B) Close-up view of the placement in panel A. The D-arm of S. cerevisiae tRNAPhe is colored black. Cm32, G34, m1G37 and SAM are indicated by the red dotted circles and black line with labels, respectively.
Figure 7.
Figure 7.
Activity of the A26P mutant. (A) Close-up view of the peripheral structure of α helix (α2) and SAM in Trm7. The residues, S25 A26 and K28 are shown as stick models (cyan). SAM is illustrated as a stick model. (B) About 12% SDS-PAGE of purified A26P mutant. The gel was stained with Coomassie Brilliant Blue. (C) Relative methyl-transfer activities of the wild-type Trm7–Trm734 (WT) and A26P mutant. The initial velocity of the WT for tRNAPhe transcript is expressed as 100.0%.

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References

    1. Boccaletto P., Machnicka M.A., Purta E., Piatkowski P., Baginski B., Wirecki T.K., de Crécy-Lagard V., Ross R., Limbach P.A., Kotter A. et al. . MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res. 2018; 46:D303–D307. - PMC - PubMed
    1. Lorenz C., Lünse C.E., Mörl M. tRNA modifications: impact on structure and thermal adaptation. Biomolecules. 2017; 7:E35. - PMC - PubMed
    1. Väre V.Y., Eruysal E.R., Narendran A., Sarachan K.L., Agris P.F. Chemical and conformational diversity of modified nucleosides affects tRNA structure and function. Biomolecules. 2017; 7:E29. - PMC - PubMed
    1. Hori H. Methylated nucleosides in tRNA and tRNA methyltransferases. Front. Genet. 2014; 5:144. - PMC - PubMed
    1. Kawai G., Yamamoto Y., Kamimura T., Masegi T., Sekine M., Hata T., Iimori T., Watanabe T., Miyazawa T., Yokoyama S. Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2′-hydroxyl group. Biochemistry. 1992; 43:1040–1046. - PubMed
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