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The selective tRNA aminoacylation mechanism based on a single G•U pair

An Author Correction to this article was published on 20 November 2018

This article has been updated

Abstract

Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNAAla selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3′-CCA region. Thus, the 3′-CCA region of tRNAAla with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.

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Figure 1: Structure of AlaRS in complex with tRNAAla.
Figure 2: Major differences between AlaR•tRNAAla/GU and AlaR•tRNAAla/AU.
Figure 3: Watson–Crick pair exclusion.
Figure 4: The tRNAAla acceptor stem is widened by the clamp.
Figure 5: Alanylation assays of tRNAAla with AlaRS.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors for AlaRS complexed with its cognate tRNAAla with G3•U70 and a variant with A3•U70 have been deposited in the Protein Data Bank (PDB) under accession codes 3WQY and 3WQZ, respectively.

Change history

  • 20 November 2018

    In Fig. 1b of this Article, a U was inadvertently inserted after G15 in the D loop. The original Article has not been corrected.

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Acknowledgements

We are grateful to the staff of the beam line AR-NW12A at the Photon Factory and the beam lines BL32XU and BL41XU at SPring-8 for their assistance during data collection. This work was supported by the Targeted Proteins Research Program, and the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also supported by a Grant-in-Aid for Scientific Research (No. 20247008) from the Japan Society for the Promotion of Science and MEXT, and the Global COE Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from MEXT. Research support was also provided by National Institutes of Health grants GM015539 and GM023562 to P.S. and NS085092 to X.-L.Y.

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M.N., S.S. and S.Y. designed the research. M.N. and S.S. performed the structural analysis. M.N., Y.E.C., M.G., X.-L.Y., H.G., and Y.-M.H. performed the enzyme kinetics analyses. M.N., S.S., P.S., and S.Y. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Shigeyuki Yokoyama.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Conformational differences between the tRNA-bound and tRNA-free subunits of A. fulgidus AlaRS.

a, Rotation of the C-terminal domain. The C-terminal domain of subunit A is inclined 28° towards the outer corner of the L-shaped tRNA, relative to subunit B, and thus the globular subdomain stacks with the outer corner of the tRNA. b, c, Mid2 reorientation. α13 is straight with tRNAAla (b) and kinked without tRNAAla (c).

Extended Data Figure 2 The positions of tRNAs relative to the aminoacylation domains of AlaRS and other class II aaRSs depend on the specific spatial arrangements of their tRNA-binding domain(s).

ad, Class II aaRS dimers bound to tRNAs. A. fulgidus AlaRS, yeast AspRS, E. coli ThrRS, and T. thermophilus SerRS are shown as surface models, with their cognate tRNAs displayed as tube models. For each aaRS, one of the two tRNA-binding aminoacylation domains (ADs) and its bound tRNA are coloured green and yellow, respectively, whereas the other AD and its bound tRNA are coloured light green and grey, respectively. In each of the aaRSs, except for A. fulgidus AlaRS, the tRNA-binding domain (RBD) corresponding to the green AD is coloured red, and that of the other subunit is coloured raspberry. A. fulgidus AlaRS has two RBDs (RBD1 and RBD2), coloured in the same manner as the RBDs of the other aaRSs. e, The position of tRNAAla relative to the AD of AlaRS (displayed as yellow and green surface models, respectively) is completely different from the well conserved positions of tRNAAsp, tRNAThr, tRNAPro, tRNASer, tRNAPhe, tRNAPyl, and tRNACys (brown tube models) shown in the same positions relative to the ADs of AspRS32, ThrRS53, ProRS54, SerRS33, PheRS55, PylRS56, and SepRS57, respectively (PDB IDs: 1ASY, 1QF6, 1H4Q, 1SER, 1EIY, 2ZNI and 2DU3, respectively).

Extended Data Figure 3 Summary of tRNA–protein interactions.

a, Schematics of tRNA-protein interactions. The tRNA backbones and bases are coloured as indicated. The G3•U70 base pair and the anticodon bases are highlighted by red and pink squares, respectively. The hydrogen bonds between the nucleotides and the AlaRS main chain are indicated with blue arrows. Those between the nucleotides and the AlaRS side chains are depicted with magenta arrows. The stacking interactions are indicated with black dashed lines. b, The tRNAAla interaction with the α11 helix. The α11 helix interacts with the backbone phosphate groups of the 3′ half of the acceptor stem. The side chains of Tyr 364 and Arg 371 hydrogen bond with C72 and C71, respectively. The side chain of Arg 375 hydrogen bonds with U70. c, Interactions between the tRNAAla outer corner region and the globular subdomain. The conserved Gly-rich segment 870KGSGGGR878 stacks with the hinge region of the T- and D-loops. The side chain of Arg 876 stacks and hydrogen bonds with the base and the backbone phosphate group, respectively, of G20. The side chain of Asn 823 hydrogen bonds with the backbone phosphate group of G20. d, The tRNAAla interactions with the α13 helix. The α13 helix interacts with the backbone phosphate groups of the D arm (U12-C13-A14-G15) and that of U70. The side chain of Thr 422 in α13 hydrogen bonds with U70. The side chains of Arg 428 and Arg 432 hydrogen bond with C13. The side chain of Arg 436 hydrogen bonds with A14 and G15.

Extended Data Figure 4 Nucleotide interactions with the conserved residues of AlaRS.

a, b, |FoFc| omit electron density maps contoured at 3σ, superimposed on the refined models of G3•U70 (a) and A3•U70 (b). cg, The interactions of G1•C72 (c), G2•C71 (d), C4•G69 (e), U5•A68 (f), and A73 (g) with AlaRS. The amino-acid side chains and the nucleotides are shown as grey and beige stick models, respectively. Hydrogen bonds are indicated with dashed lines. h, A73 stacking with G1 and C72. i, Alignments of the AlaRS sequences. A total of 249 AlaRS sequences were initially aligned with the ClustalW program58, and then were manually adjusted based on the structural information. The sequences of the A. fulgidus, P. horikoshii, Homo sapiens, E. coli, and A. aeolicus AlaRSs are shown. The highly-conserved residues are boxed in red. The residues that are involved the acceptor stem interactions in A. fulgidus AlaRS are boxed in blue. Secondary structure information is shown above the A. fulgidus sequence. The full-sequence alignments were reported previously28.

Extended Data Figure 5 The routes of the CCA regions of tRNAAla/GU and tRNAAla/AU.

ac, tRNAAla/GU. df, tRNAAla/AU. tRNA is shown in stick models (a, d), cartoon models (b, e), and surface models (c, f). The |FoFc| omit electron density maps contoured at 3σ are superimposed on the refined models of the CCA regions of tRNAAla/GU (a) and tRNAAla/AU (d). The CCA region of tRNAAla/GU is in the reactive route (b, c), while that of tRNAAla/AU is in the non-reactive route (e, f). The CCA region runs under the linker (b, c, e, f). g, h, Heterologous superimpositions of the |2FoFc| electron density maps contoured at 1σ of tRNAAla/AU (cyan, g) and tRNAAla/GU (pink, h) on the stick models of the structures of the acceptor stems and the CCA regions of tRNAAla/GU (pink, g) and tRNAAla/AU (cyan, h), respectively, which clearly demonstrate the conformational difference between the two tRNAs. i, The |FoFc| omit electron density maps for tRNAAla/AU, contoured at 2.5σ, superimposed on the refined models of the C74 s of tRNAAla/GU (pink) and tRNAAla/AU (cyan). j, k, The similarity in the spatial positions between E220/193GGG195 of A. fulgidus AlaRS (j) and D174 of E. coli AlaRS (k), marked in red on the surface models of the aminoacylation and tRNA-recognition domains with the tube models of tRNAAla (the crystal structure and the docking model, respectively). l, Misorientation of the CCA region of tRNAAla/WC on the D450A mutant of AlaRS.

Extended Data Figure 6 Major-groove widening of the tRNAAla acceptor stem.

a, The phosphate–phosphate distances of G1–C66, C2–C65, G3–G64, G4–C63, A5–C62, and U6–C61 in tRNAPhe (refs. 59, 60). b, The phosphate–phosphate distances of G1–C66, G2–C65, G3–G64, C4–C63, U5–C62, and C6–C61 in the wild-type tRNAAla (tRNAAla/GU) complexed with AlaRS. The phosphate–phosphate distances between G1–C66, G2–C65, and G3–G64 across the major groove of tRNAAla/GU are extended by 6.3, 2.9, and 0.9 Å, respectively, as compared to those of tRNAPhe. Thus, AlaRS clearly widens the major groove of the acceptor stem of tRNAAla/GU. c, The phosphate–phosphate distances of G1–C66, G2–C65, A3–G64, C4–C63, U5–C62, and C6–C61 in the variant tRNAAla/AU complexed with AlaRS. The major-groove of the tRNAAla/AU acceptor stem is slightly wider than that in the wild-type complex structure. Therefore, the replacement of G3•U70 by A3•U70 does not seem to impair the major-groove widening by AlaRS. d, The phosphate–phosphate distances of U1–U66, C2–G65, C3–C64, G4–C63, U5–C62, and G6–C61 in tRNAAsp bound to AspRS32 (PDB ID: 1ASY). e, The phosphate–phosphate distances of G1–U66, C2–U65, C3–A64, G4–U63, A5–C62, and U6–C61 in tRNAThr bound to ThrRS53 (PDB ID: 1QF6). f, The phosphate–phosphate distances of G1–U66, G2–C65, C3–C64, C4–C63, C5–C62, and C6–C61 in tRNAGlu bound to GluRS61 (PDB ID: 1G59). g, The AlaRS-induced widening of the tRNAAla acceptor stem. Without widening, AlaRS clashes with A67-G68. h, Widening of the acceptor stems of tRNAAla/GU (pink tube models) and tRNAAla/AU (cyan tube models).

Extended Data Figure 7 Torsion angles of nucleotides in the acceptor stems of tRNAAla/GU and tRNAAla/AU.

a, b, Tables for torsion angles of nucleotides in the acceptor stems of tRNAAla/GU (a) and tRNAAla/AU (b). c, Conformational changes of G3 and G69 relative to the A-form RNAs.

Extended Data Figure 8 tRNAAla/AU inhibits aminoacylation of human tRNAAla/GU by human AlaRS.

In vitro transcribed human tRNAAla/AU was added to aminoacylation reactions containing 2 μM human tRNAAla and 50 nM human AlaRS, in pH 6.0 reaction buffer at room temperature. The dose-dependent inhibition of aminoacylation of tRNAAla/GU by excess tRNAAla/AU shows that the ultimate discrimination against A3•U70 is via kcat.

Extended Data Table 1 Data collection and refinement statistics (molecular replacement)
Extended Data Table 2 Kinetics of wild-type and mutant AlaRSs

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Naganuma, M., Sekine, Si., Chong, Y. et al. The selective tRNA aminoacylation mechanism based on a single G•U pair. Nature 510, 507–511 (2014). https://doi.org/10.1038/nature13440

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