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Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

The anticodon modifications of transfer RNAs (tRNAs) finetune the codon recognition on the ribosome for accurate translation. Bacteria and archaea utilize the modified cytidines, lysidine (L) and agmatidine (agm2C), respectively, in the anticodon of tRNAIle to decipher AUA codon. L and agm2C contain long side chains with polar termini, but their functions remain elusive. Here we report the cryogenic electron microscopy structures of tRNAsIle recognizing the AUA codon on the ribosome. Both modifications interact with the third adenine of the codon via a unique C–A geometry. The side chains extend toward 3′ direction of the mRNA, and the polar termini form hydrogen bonds with 2′-OH of the residue 3′-adjacent to the AUA codon. Biochemical analyses demonstrated that AUA decoding is facilitated by the additional interaction between the polar termini of the modified cytidines and 2′-OH of the fourth mRNA residue. We also visualized cyclic N6-threonylcarbamoyladenosine (ct6A), another tRNA modification, and revealed a molecular basis how ct6A contributes to efficient decoding.

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Fig. 1: tRNA modifications in the anticodon regions and the cryo-EM structure of the 70S ribosome bound to tRNAIle2 recognizing the AUA codon.
Fig. 2: Structural basis of AUA decoding by C*34 in tRNAIle2.
Fig. 3: Biochemical characterization of AUA decoding by C*34 with synthetic mRNAs.
Fig. 4: Structural characterization of hypermodifications at position 37.

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Data availability

Publicly available datasets from Protein Data Bank (7K00, 4V8N and 4V5R) were used for atomic model building and comparison. Cryo-EM maps and atomic coordinates of the reported structures were deposited in Electron Microscopy Data Bank (EMDB) and Protein Data Bank, respectively, with the following accession codes: EMD-35001 and 8HSP (EctRNAIle2 with t6A37); EMD-35022 and 8HU1 (EctRNAIle2 with ct6A37); EMD-35020 and 8HTZ (HmtRNAIle2). Source data are provided with this paper.

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Acknowledgements

We are grateful to the members of the Suzuki laboratory, in particular, Y. Sakaguchi, for technical support and insightful discussion. Special thanks are due to M. Gagnon and M. Rybak (UTMB) for sharing unpublished data with us and productive discussion. All cryo-EM data in this study were collected at the cryo-EM facility of the RIKEN Center for Biosystems Dynamics Research (Yokohama). We thank T. Uchikubo-Kamo and R. Akasaka for their help with cryo-EM data collection and analysis. Radioisotope experiments were carried out with the support of the Isotope Science Center, Univ. of Tokyo. This work was supported by a grant in aid for scientific research from MEXT and JSPS (26113003, 26220205, and 18H05272 to T.S.; 26116003 and 25660053 to A.N.; 18K05430 to K.M.; 20J00947 to K.I.; 23KJ0409 to N.A.), AMED (JP21am0101115 to T.Y.; JP223fa627001 to T.S.; JP21am0101082 to M.S.), RIKEN (Pioneering project ‘Biology of Intracellular Environments’ and BDR Structural Cell Biology Project to M.S.) and JST (ERATO, JPMJER2002 to T.S.).

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Authors and Affiliations

  1. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan

    Naho Akiyama, Kensuke Ishiguro, Kenjyo Miyauchi, Asuteka Nagao & Tsutomu Suzuki

  2. Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan

    Kensuke Ishiguro, Takeshi Yokoyama & Mikako Shirouzu

  3. Graduate School of Life Sciences, Tohoku University, Sendai, Japan

    Takeshi Yokoyama

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Contributions

N.A. prepared all materials, performed biochemical experiments assisted by A.N. and K.M. and conducted cryo-EM analyses supported by K.I., T.Y. and M.S. N.A. and T.S. wrote the manuscript. All authors discussed the results. T.S. designed and supervised the project.

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Correspondence to Tsutomu Suzuki.

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Extended data

Extended Data Fig. 1 Mass spectrometric characterization of the native tRNAs used in this study.

(a) Secondary structures of E. coli (left panel) and H. marismortui (right panel) tRNAIle2 with post-transcriptional modifications. Abbreviations are as follows: s4U, 4-thiouridine; Gm, 2’-O-methylguanosine; D, dihydrouridine; L, lysidine; t6A, N6-threonylcarbamoyladenosine; Ψ, pseudouridine; m7G, 7-methylguanosine; acp3U, 3-(3-amino-3-carboxypropyl)uridine; T, 5-methyluridine; G+, archaeosine; m2,2G, N2, N2-dimethylguanosine; agm2C, agmatidine; m5C, 5-methylcytidine; m1Ψ, 1-methylpseudouridine; Cm, 2’-O-methylcytidine; and m1I, 1-methylinosine. The sequence colored in red corresponds to the RNase T1 fragment containing the anticodon region. (b) PAGE analysis of tRNAIle2 isolated by RCC. Total RNAs are used as size markers. A band of impurity is asterisked. Isolation and electrophoresis of tRNA were repeated at least three times, yielding similar results. The representative gel image is shown. (c) Mass spectrometric analyses of tRNAIle2 isolated from the E. coli ΔtcdA strain (left panels) and H. marismortui (right panels). Extracted-ion chromatograms (XICs) of C34-containing fragments (top panels) and L34- or agm2C-containing fragments (bottom panels) of tRNAIle2 digested by RNase T1. The sequences of the detected fragments with m/z values and charge states are indicated on the right.

Source data

Extended Data Fig. 2 In vitro reconstitution of ct6A37 in E. coli tRNAIle2.

(a) Schematic depiction of ct6A formation catalyzed by TcdA in the presence of ATP. (b) Mass spectrometric analyses of E. coli tRNAIle2 before (left panels) and after (right panels) ct6A reconstitution. XICs of t6A37-containing fragments (upper panels) and ct6A37-containing fragments (lower panels) of E. coli tRNAIle2 digested by RNase A and treated with BAP. The sequences of the detected fragments with m/z values and charge states are indicated on the right.

Extended Data Fig. 3 Structural analyses of 70 S ribosome complexed with A- and P-site tRNAs.

(a) Image processing of the ribosome complexes with E. coli or H. marismortui tRNAIle2 at the A-site. Auto-picked particle images were subjected to 2D classification in order to remove low quality particles and auto-refinement. After 3D classification, the subclasses of 70 S ribosomes with P-site occupancy were pooled for 3D refinement; this pool included all particles potentially bound by A-site tRNAIle2. Particles with A-site density were extracted by focused classification using an A-site mask and used to generate the final map. The overall cryo-EM maps are shown and colored according to the local resolution. (b, c) Fourier shell correlation (FSC) curves of the complexes (b) and models vs. cryo-EM maps (c). (d) Isolated densities of tRNA and mRNA colored according to the local resolution. From left to right, EctRNAIle2 + t6A, EctRNAIle2+ct6A, HmtRNAIle2.

Extended Data Fig. 4 Cryo-EM structure of H. marismortui tRNAIle2.

(a) Atomic model of the E. coli 70 S ribosome (23 S; light green, 16 S; olive, r-proteins; yellow) bound with E. coli tRNAGlu at the P-site (magenta) and H. marismortui tRNAIle2 at the A-site (orange). The molecular surface is displayed on each tRNA model. (b) Codon-anticodon duplex of H. marismortui tRNAIle2 (orange) and the AUA codon (gray) in the decoding center of 16 S rRNA (olive). A1913 (light green) in Helix 69 of 23 S rRNA and S12 (yellow) are displayed. (c) The architecture of the decoding center around L34 (left panel) or agm2C34 (right panel). The backbone of mRNA third residue is coordinated with a potassium ion bound to C518 and G530 of 16 S rRNA, and Pro45 of S12.

Extended Data Fig. 5 Codon-anticodon interactions at the P-site.

The codon-anticodon duplex of E. coli tRNAGlu (purple) and the GAG codon (gray) at the P-site (left panel) in the complex with E. coli tRNAIle2(+t6A). Base pairing geometry of mnm5s2U34 recognizing G3 of the GAG codon. Post-processed maps (contoured at level 0.02) are superimposed on the model structure. The geometry of the minor groove-shifted wobble base pair is identical to that at the A-site described previously26. The density of the methylaminomethyl (mnm) group at the C5 atom is visible at this contour level.

Extended Data Fig. 6 Comparison of base pairing geometries between C34-G3 (gray), L34-A3 (blue), and agm2C-A3 (orange).

When C*34-A3 pairs and C34-G3 canonical Watson-Crick pair (PDB ID: 4V5R) are superimposed by the mRNA base, the cytosine base of C*34 is displaced to its minor groove by 2.7–2.8 Å.

Extended Data Fig. 7 Fitting of the C*34 side chain into the cleft formed by rRNA and mRNA.

Solvent-excluded surface models showing the structural complementarity of the long side chains of L34 (left panel) and agm2C (right panel), and the cleft formed by rRNA residues and the mRNA strand. A large area of van der Waals contacts between C*34 and rRNA residues and the mRNA is clearly visible, especially in the agm2C model.

Extended Data Fig. 8 Conformational restriction of the hydantoin ring of ct6A37.

(a) Ab initio conformational energies in the ct6A nucleoside for the rigid scan over the dihedral angle η (N1-C6-N6-C13)30. \(\eta =\)21.7° (suggested in our model) is indicated by red line. Twisted rotamers with different η showing the minimum energies are shown by blue line. (b) Atomic model of ct6A37 and the neighboring residues based on our cryo-EM map (\(\eta =\)21.7°). (c-f) Simulated atomic models of ct6A37 with different η showing the minimum energies suggested by quantum calculations30 in (a). ct6A37 was modeled with the following dihedral angles η of the hydantoin ring and the adenine base; (c) \(\eta =\)67.3°; (d) \(\eta =\)127.3°; (e) \(\eta =\)-52.7° (consistent with the single crystal structure30); (f) \(\eta =\)-122.7°. In these models, the hydantoin ring shows an apparent steric clash with neighboring nucleotides in the codon-anticodon helix.

Extended Data Fig. 9 Comparison of previous and current models of agm2C34 recognizing A3 of the AUA codon.

The C-A geometry is consistent between the previous model (yellow, PDB ID: 4V8N) and the current model (orange). However, the orientation of the agmatinyl moiety is markedly different. In our structure, the clear density of agm2C34 enables to fully model its long side chain with a terminal guanidino group.

Extended Data Fig. 10 Alternative structural model of L34 recognizing the fourth mRNA residue.

(a) Alternative interactions of L34 recognizing the AUA codon at the A-site. The terminal amino group of L34 has a potential to form an H-bond with 2’-OH of the fourth mRNA residue (N4) considering 2’-F substitution maintained AUA decoding efficiency. (b) Simulated atomic model of L34 rotamer in which the amino group of L34 serves as a hydrogen donor.

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Source data

Source Data Fig. 3

Statistical source data for Fig. 3c,e.

Source Data Extended Data Fig. 1

Unprocessed gel image.

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Akiyama, N., Ishiguro, K., Yokoyama, T. et al. Structural insights into the decoding capability of isoleucine tRNAs with lysidine and agmatidine. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01238-1

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