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The structural basis of transfer RNA mimicry and conformational plasticity by a viral RNA

Abstract

RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles, and this can be conferred by a complex three-dimensional structure. Such multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states1. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. To address this gap it is useful to study examples from single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3′ end of the turnip yellow mosaic virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic (g)RNA2,3,4, but also interacts with other structures in the 3′ untranslated region of the gRNA5, contains the promoter for negative-strand synthesis, and influences several infection-critical processes6. TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is ‘two faced’: one face closely mimics tRNA and drives aminoacylation, the other face diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes.

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Figure 1: Function and structure of the TYMV TLS.
Figure 2: Structural differences between tRNA and the TLS.
Figure 3: tRNA mimicry and AARS binding.
Figure 4: Binding of tRNA and TLS to ribosomes.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession number 4P5J.

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Acknowledgements

We thank H. Noller for the gift of 70S ribosomes. We thank I. Tinoco Jr, C. Musselman and T. Dreher for critical reading of this manuscript. The University of Colorado (UC) Denver X-ray Facility is supported by UC Cancer Center Support Grant P30CA046934. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences of the US Department of Energy under contract #DE-AC02-05CH11231. J.S.K. is supported by grants GM081346 and GM097333 from the National Institutes of Health and is an Early Career Scientist of the Howard Hughes Medical Institute.

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Contributions

J.A.H. and G.M.R. designed the crystallization RNAs and identified initial crystals. T.M.C. and D.A.C. improved and grew the crystals. Crystals were harvested by T.M.C., who also solved, built and refined the structure. J.C.N. collected and processed synchrotron diffraction data. G.M.R. conducted the ribosome binding experiments. J.S.K. provided overall supervision and guidance, and together with T.M.C. and D.A.C. wrote the manuscript.

Corresponding author

Correspondence to Jeffrey S. Kieft.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequences and structures of RNAs.

Top left, sequence and secondary structure of the complete TYMV TLS and the UPD (green dashed box). The UPD is just upstream of the UUAG sequence that is important for stabilizing the L-shaped structure and the UPD is known to be able to pack against the TLS5,11. Interestingly, the stop codon for the Coat protein is within the UPD (magenta). Right and bottom, sequences and secondary structures of all additional RNAs used in ribosome binding assays or discussed in the text. Yellow highlights indicate the location of mutation.

Extended Data Figure 2 Representative electron density and bound trivalent ions.

a, Unbiased, density-modified electron density from SAD phasing using data to 2.5 Å (grey mesh, 2σ), superimposed on the final model. The T loop and part of the D loop is shown. For simplicity, the density and structure of water and ions is not shown. b, Final 2Fo − Fc electron density map after model building and refinement to 1.99 Å (2σ). c, Structure with the location of 12 iridium (III) hexammine ions. Although many of these hexammine binding sites may also be Mg2+ binding sites important for stabilizing the fold, the trivalent hexammine was present at 8 mM and thus many weaker Mg2+ binding sites could have been occupied. For this reason, and because there is not a one-to-one correlation of Mg2+ binding sites and trivalent hexammine sites, we do not make conclusions about Mg2+ binding on the basis of this structure.

Extended Data Figure 3 Topologies and three-dimensional structures of tRNA and the TYMV TLS.

a, Top, the topology of a canonical tRNA is shown in rainbow colours with the 5′ end in blue and the 3′ end in red. The attached amino acid is shown (labelled ‘aa’ or Val) and structural features are labelled: AC, anticodon loop; D, D loop, T, T loop; V, variable loop. The 5′ and 3′ ends of the RNA are shown. Bottom, ribbon representation of the backbone of tRNAPhe coloured roughly to match the cartoon diagram. b, Same as a, but for the TYMV TLS. The location of the UPD (grey dashed box) and gRNA (grey dashed line connected to the 5′ end) are shown on the top diagram.

Extended Data Figure 4 Assignment of bases to the syn conformation.

a, Nucleotide G4, which forms the long-range base pair with C76 in the pseudoknot, is in a syn conformation. Top, placement of the base into an anti conformation results in positive and negative density (green and red, respectively) in the Fo − Fc map (left, contoured at 3σ), and the 2Fo Fc map (right) shows the base is incorrectly placed (blue density, contoured at 1.5σ). In contrast, placement of the base into the syn conformation (bottom) results in a flat Fo − Fc map (left, contoured at 3σ) and a good fit to the 2Fo− Fc map (right, blue density contoured at 1.5σ). Base A11 is also in a syn conformation; the same analysis was performed to verify this (data not shown). b, 2Fo − Fc map surrounding bases A3–C5. The C4′–C5′ bond of G4 is best modelled in the trans conformation.

Extended Data Figure 5 Effect of breaking the linchpin interaction.

a, Small-angle X-ray scattering (SAXS) analysis of TYMV TLS RNAs, adapted with permission from ref. 5. Left, ab initio SAXS reconstruction of the shape of the TLS when the 5′ sequence that interacts with the pseudoknot (Fig. 2) is present. The RNA forms an L shape overall, illustrated by the black bars (stabilizing long-range interaction in grey). When these 5′ nucleotides are removed (right), the L shape is lost and the RNA becomes more extended. b, Hydroxyl radical probing of several TYMV TLS RNAs that indicate the effect of disrupting the long-range interaction, adapted with permission from refs 5, 11. Green and red indicate protection from cleavage by radicals and enhanced cleavage by radicals, respectively. Overall, the presence of green and red indicate tightly folded RNA. When the 5′ nucleotides that form the long-range interaction are present, the RNA stably folds (TYMV UUAG, left). Removal of the 5′ nucleotides destabilizes the fold (TYMV 0G, right). The presence of just G4 on the 5′ end partially stabilizes the RNA fold (TYMV 1G, middle), confirming its importance in folding and also indicating that the nucleotides adjacent to G4 further stabilize the fold.

Extended Data Figure 6 T loop and acceptor stems of the tRNA and TLS, and elongation factor binding.

a, Superimposed structures of the TLS T loop (red) and part of the D loop (cyan) with the analogous structures in tRNA (grey). TLS bases A11 and A12 are shown; these bases match the interactions formed by analogous bases in tRNA. In the TLS, A11 is in a syn conformation, but the matching base in tRNA is not. This may be due to local differences in the backbone conformation. b, Superimposed structures of the TLS T loop (red) and pseudoknot (blue) with the T loop and acceptor stem elements in a tRNA (grey). View is from the ‘top’ of the molecule, down the axis of the D and AC stems. c, Top, the structure of the T loop (red) and acceptor stem pseudoknot (blue) in the TLS crystal structure. Bottom, structure of these elements isolated from the rest of the TLS and solved by NMR (Protein Data Bank accession 1A60)43. d, Superposition of the TLS structure (red) onto the tRNA (cyan) of a tRNAPhe bound to EF-Tu (yellow), the bacterial homologue of eEF1A (Protein Data Bank accession 1TTT)44. Binding is probably facilitated by the fact that the RNA backbone conformation of the TLS pseudoknot and T stem/loop matches that of a tRNA.

Extended Data Figure 7 The ‘two-faced’ architecture of the TYMV TLS and connection with the UPD.

Several views of the TLS (red) superimposed on tRNAPhe (cyan)21 are shown, rotated 90° relative to each other. The dashed line bisects the structure into its two faces. The backbones are very similar on the tRNA-like face, but differ on the divergent face. Locations where the two structures diverge most markedly are shaded grey. The 5′ end of the TLS, where the UPD connects, is indicated.

Extended Data Figure 8 The AC loop: structures and crystal packing.

a, Structure of the AC loop of tRNAPhe, solved to 1.93 Å (ref. 21). The loop is coloured to reflect relative B factors, with red as the highest and blue as the lowest. b, Structure of the AC loop of the TYMV TLS, coloured identically to a. The asterisk marks the C30 base that was mutated to G to enhance crystallization. This was the only mutation made to the TLS for crystallization and does not inhibit aminoacylation32. Overall, the loop structures are similar and both have high crystallographic B factors compared with other parts of the structures, a common feature of tRNAs. There is no evidence that the TYMV TLS AC loop is post-transcriptionally modified, yet it has structural features and conformal flexibility similar to the AC loop of a tRNA (which is often modified; Fig. 2a). c, Crystal packing involving the AC loop of the TYMV TLS. Two interacting copies of the RNA are shown in red and magenta, with the C30G mutation in yellow. This mutation, although not appearing to alter the overall AC-loop structure compared to a tRNA, induces intermolecular base pairing in the crystal (pattern shown to the right), suggesting why this mutation aided crystallization. d, Crystal packing of the 3′ CCA of the TLS (red, labelled) against an adjacent molecule (magenta) probably causes the CCA to adopt a folded-back conformation.

Extended Data Figure 9 Models of protein binding to the TLS and the location of the UPD.

a, Model of the TLS (red, backbone ribbon shown) on the valine of AARS (green; Protein Data Bank accession 1GAX), similar to Fig. 3b, but viewed from the top and with the tRNAVal not shown. The location of the UPD directly 5′ of and against the TLS is shown as a grey oval. The viral genomic RNA is 5′ of the UPD. Note that the strategy used by the TYMV TLS to interact with this protein is probably very different from that used by the TLSs that are histidylated or tyrosylated, which are very different in terms of their secondary structure and fold6,9. b, Same as a, but with the TLS modelled onto the bacterial homologue of eEF1A (EF-Tu) as in Extended Data Fig. 6. tRNAPhe is not shown. In both complexes, the location of the 5′ end, the UPD, and viral genome would not interfere with protein binding. This would not be true if the TLS had a tRNA-like topology with the 5′ end paired to the 3′ end.

Extended Data Table 1 Crystallographic data collection, phasing and refinement statistics

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Colussi, T., Costantino, D., Hammond, J. et al. The structural basis of transfer RNA mimicry and conformational plasticity by a viral RNA. Nature 511, 366–369 (2014). https://doi.org/10.1038/nature13378

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