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

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. This 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. Examples to address this gap are found in 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 RNA (gRNA)2-4, but also interacts with other structures in the gRNA's 3′ untranslated region5, contains the promoter for negative strand synthesis, and influences several infection-critical processes6. This 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.

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.
Keywords turnip yellow mosaic virus (TYMV); RNA structure; x-ray crystallography; ribosome binding; pseudoknot The TYMV TLS RNA ('the TLS') is a tRNA mimic, a subject of broad biological and evolutionary importance 7 , underscored by examples linked to disease [8][9][10] . Like tRNA, the aminoacylated TLS binds to eukaryotic elongation factor 1A (eEF1A) and is a substrate for tRNA-modifying enzymes 6 . These activities and other data suggest a tRNA-like structure [11][12][13][14][15][16] . However, the TLS' topology differs from tRNA, mandated by its location on the 3′ end of the gRNA (Fig. 1b, Extended Data Fig. 1). In addition to affecting many viral processes [17][18][19] , the TLS may regulate the activities of ribosomes and replicases on the gRNA 6,20 . This function could be conferred by the TLS' ability to readily transition between folded and unfolded states. Simple tRNA mimicry is insufficient to explain these phenomena; while tRNAs flex while transiting through the ribosome they do not unfold and refold. To explore the paradigms of tRNA mimicry and RNA structural and functional plasticity, we solved the structure of the TYMV TLS RNA by X-ray crystallography to 2.0 Å resolution (Fig. 1c, Extended Data Fig. 2), comparable to the highest-resolution structure of free tRNA, used here for comparison (1.93 Å) 21 .
The TLS assumes the classic L-shaped tRNA conformation (Fig. 1d), but achieves this in a way that diverges from tRNA and from predictions 13,22 . Compared to tRNA, the topology (Fig. 1b, Extended Data Fig. 3) and the intramolecular interactions that form the structure diverge from a tRNA's ( Fig. 2a). Although the TLS pseudoknot (the first recognized RNA pseudoknot 14 ) is in the position of the tRNA's acceptor stem and elements analogous to the D-loop, T-loop, and V-loop are positioned as in tRNA, their interactions are not tRNA-like. In the elbow region of tRNA, the V-loop interacts with the D-stem, stabilizing the L-shaped tRNA structure (Fig. 2b). In contrast, the TLS' V-loop bases point away from the D-stem to interact with the 5′ end and pseudoknot of the TLS (Fig. 2b). G4 adopts a syn conformation (Extended Data Fig. 4), forming a long-range base pair with C76 in a loop of the pseudoknot. The unexpected G4-C76 base-pair is stabilized by stacking of A3 and the Vloop base A42 on either side. V-loop bases A42-U44 continuously stack to reach from the G4-C76 pair to A15 of the D-loop, structurally linking the pair to the elbow region. These interactions explain the observation that removing 5′-UUAG sequence from the 5′ end of the TLS (including G4) destabilizes the L-shape conformation and elbow structure (Extended Data Fig. 5) 5,11 . Although historically not recognized as part of the minimal TLS, these 5′ nucleotides form a 'linchpin' interaction that stabilizes the global structure and this suggests why their presence increases aminoacylation efficiency (Fig. 2a, Extended Data Fig. 6).
The structural features of the elbow region require that the TLS D-loop be in a different conformation than tRNA's. In the TLS, the D-loop contains a tight bend that allows A15 to reach across the helix to stack on U44 in the V-loop (Fig. 2c), while U13 and U14 lie against the end of the T-loop. No analogous bases or interactions are found in tRNA. Despite this, the T-loops of the TLS and tRNA are structurally identical (Extended Data Fig. 6). D-loop bases G12 and A11 dock into the T-loop almost identically to the analogous bases of tRNA, although A11 is in a syn conformation.
The TLS' stabilizing intramolecular interactions show how it can adopt different folded states to potentially organize infection-important activities, achieving structural and functional plasticity. Disruption of the 'linchpin' would lead to a loss of the L-shape fold and a propagated loss of interactions extending from the V-loop to the D/T-loop interface. This effect is observed when the base pair and adjacent nucleotide that stack on and stabilize this pair are eliminated by truncating the TLS from the 5′ end 5,11 (Extended Data Fig. 5). This disruption could be induced by loading of the virally-encoded RNA-dependent RNA polymerase (RDRP) at the 3′ end 18,23,24 . The subsequent destabilization would create a favorable template for the RDRP and effectively remove competition between the RDRP and the proteins that require the stable fold (e.g. the amino acid synthetase; AARS).
The TLS structure has two distinct 'faces'. The tRNA-deviating features are on one side of the structure, where the UPD and the gRNA connect to the TLS (Fig. 1b, Extended Data Fig. 7). The structure reveals that the UPD is positioned to interact with the TLS' 'divergent face'. The opposing side of the TLS, the 'tRNA-like face', interacts with the valine AARS when the TLS structure is modeled into a tRNA Val •AARS complex structure 25 (Fig. 3a,b). The TLS structure is accommodated by the AARS, including the acceptor stem pseudoknot, which has a different structure than shown by NMR (Extended Data Fig. 6). Like tRNA, the TLS has high crystallographic B-factors in its anticodon (AC) loop and 3′ CCA, suggesting these can readily undergo structural changes ( Fig. 3c,d, Extended Data Fig. 8). In the case of the AC loop, this is important to dock the valine-specifying identity elements in the AC loop onto the protein 26 . Modeling of the TLS structure onto an elongation factor structure also reveals an interface similar to that formed with tRNA and no obvious steric clash (Extended Data Fig. 6). Because the divergent face does not contact the AARS or eEF1A, the 5′ end of the TLS is not occluded by interaction with either protein. Thus, the UPD and viral genome do not interfere with binding (Extended Data Fig. 9), and the precise mimicry of the tRNAlike face explains how the TLS can achieve tRNA-like valylation efficiencies and eIF1A binding affinities 27 .
The TLS' interactions with AARS and eEF1A suggest it could bind to the ribosome as previously suggested 6,28 . Ribosome binding would require accommodating the entire TLS structure between the subunits, including elements that deviate from tRNA within the TYMV 3′UTR. We measured binding of TLS-containing RNAs to Thermus thermophilus 70S ribosomes, a valid model for tRNA binding assays given the interchangeability of eukaryotic and bacterial tRNAs 29 . In vitro transcribed A. thaliana tRNA Val bound to the 70S (K d =0.27 +/-0.05 nM) while a 75 nucleotide-long negative control RNA (from bacteriophage phi29 pRNA) did not (K d >1000 nM) (Fig. 4a, Extended Data Fig. 1). Mutation of the tRNA Val D-loop to disrupt the global tRNA fold resulted in a 28-fold loss of affinity (K d >7.6 +/-0.8 nM) (Fig. 4b), consistent with binding being dependent on the tRNA's global conformation. A TLS RNA containing the 5′-UUAG sequence bound with tRNA-like affinity (K d = 0.31 +/-0.07 nM), and mutation of this RNA's D-loop decreased binding 9-fold (K d >2.7 +/-0.2 nM) (Fig. 4c). Likewise, truncation of the TLS' 5′ end to abrogate the 'linchpin' interaction reduced binding ∼3-fold (K d =1.1 +/-0.3 nM) (Extended Data Fig. 5). Remarkably an RNA containing the TLS, the UUAG, and the 23 nucleotidelong UPD also bound to ribosomes (TYMV UPD; K d = 0.24 +/-0.11 nM), and binding of this RNA was reduced 100-fold by D-loop mutation (K d >24 +/-8 nM) (Fig. 4d). Thus, the folded TLS can bind the ribosome even in the context of the entire 3′ UTR and binding depends on native structure. The affinity is consistent with binding to the P site, although binding to other sites is possible. The ability of the entire TYMV 3′ UTR to dock within ribosomes may relate to its functions as a regulatory switch, a translation enhancer and a means to protect the 3′ end of the genomic RNA [17][18][19] .

General Procedures
Chemical Reagents and Synthetic DNA: General chemical reagents were all of molecular biology grade or higher. All aqueous solutions were made using diethylpyrocarbonate (DEPC)-treated milli-Q water and routinely filtered through 0.22 μm sterile filtration systems (Millipore). DNA primers were purchased from Integrated DNA Technologies and used without further purification. Nucleic acid concentrations were determined by monitoring a solution's absorbance at 260 nm using a Nanodrop UV-Vis spectrophotometer (Thermo). Iridium (III) hexammine was synthesized as described 31 .

RNA transcription
dsDNA templates for transcription were made by PCR using template plasmid DNA that contained the sequence of interest (plasmids made using standard mutagenesis methods). DNA from a 1 mL PCR reaction was used in a 5 mL in vitro transcription reaction with final concentrations of 30 mM Tris-HCl pH 8.0, 10 mM DTT, 0.1% Triton X-100, 0.1% Spermidine, 40 mM MgCl 2 , 4 mM each NTP, and T7 RNA Polymerase. The reaction was incubated at 37°C for 6 hours. Inorganic pyrophosphate was pelleted at 3000 × g for 10 minutes, followed by EtOH precipitation of the supernatant. Precipitated RNA was pelleted by centrifugation, dried, then resuspended in 8 M Urea. RNA was purified on a 10% denaturing PAGE slab gel at 40 W for 5 hours, then excised and passively eluted in DEPCtreated water overnight at 4°C. RNA was concentrated and exchanged into DEPC-treated water by ultrafiltration and stored at -20°C.

RNA crystallization and diffraction data collection
The RNA sequence used in crystallization was based on a sequence identified by in vitro selection for TYMV TLS RNAs capable of efficient valylation and contained a point mutation in the anticodon loop 32 . This RNA was prepared for crystallography in a solution containing 5 mg/mL RNA, 2.5 mM MgCl 2 , and 10 mM HEPES-KOH pH 7.5. This mixture was heated to 65°C for 3 minutes, then cooled at room temperature. After cooling, Spermidine was added to 0.5 mM. The reaction was centrifuged for 10 minutes at 13000 × g and then used in sitting-drop vapor diffusion crystallization at 4°C. 1 μL of RNA solution was combined with 2 μL of 10% MPD, 40 mM Na-Cacodylate pH 6.0, 12 mM Spermine, 80 mM NaCl, and 20 mM MgCl 2 . The well solution was 20-35% MPD. Crystals appeared and grew to full size over the course of 1-2 days. To obtain derivatized crystals for phasing, a solution matching the well solutions with the addition of 8 mM Iridium (III) hexamine was exchanged with the crystal growth solution. Crystals were harvested directly from the drops into nylon loops and flash-frozen by plunging into liquid nitrogen. Diffraction data were collected at Advanced Light Source Beamline 4.2.2 using "shutterless" collection at the Iridium L-III edge (1.0972 Å) at 100° K. For each crystal, multiple 180° datasets were collected with 0.1° oscillation images. Data were indexed, integrated, and scaled using XDS 33,34 .

Structure determination and refinement
Although data were collected and processed to 1.99 Å, only data to 2.5 Å were used for phasing. Fifteen Iridium (III) hexammine sites were identified and used in SAD phasing within the AUTOSOL function of PHENIX (overall FOM = 0.448) 35 . Scattering factors used were f′ = -11.92, f″= 10.09. Density modification using RESOLVE (solvent content set to ∼50%) led to an interpretable electron density map (Extended Data Fig. 2). Iterative rounds of model building and refinement (simulated annealing, rigid-body, B-factor refinement, phase combination using COOT 36,37 and PHENIX REFINE) led to the final model. The final model contains 84 of 86 nucleotides, 2 Mg 2+ ions, 12 Iridium (III) hexammine ions, one Spermine molecule and 146 water molecules. Crystal diffraction data, phasing, and refinement statistics are contained in Extended Data Table 1. For further analysis of the structure was completed using MolProbity 38,39 . Summary of the output: Clashscore = 12.52; Probably wrong sugar puckers: 2; Bad backbone conformations: 7; Bad bonds: 1; Bad angles: 1. Areas of concern were examined in the structure and generally fell within areas of the structure with unusual conformations, but the density and model agreed well in these regions.

Mutagenesis for ribosome binding
Mutations to the DNA templates were made using a PCR-based site-directed mutagenesis protocol (Agilent) with primers designed to modify the D-loop nucleotides. The nucleotides comprising the D-loops of tRNA Val , TYMV-UUAG TLS, and TYMV-UPD TLS were replaced with stable UUCG tetraloop sequences. For tRNA, the primer sequence was 5′-GGG TGG TGT ACT TCG GAC GCT AGT CTC-3′. The UPD primer had the sequence 5′-CTT TAA AAT CGT TAG CTC GCT TCG GCG AGG TCT GTC CCC-3′. The UUAG primer sequence was 5′-CCG TCT TAG CTC GCT TCG GCG AGG TCT GTC CCC-3′.

70S Ribosome Purification
Preparation of 70S ribosomes was done by the Noller lab (UC Santa Cruz) as described 40 .

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Filter Binding
The filter binding protocol used was modified from previously published methods 41,42 . 50 μL reactions contained 25mM Tris-HCl, 50mM KCl, 10mM MgCl 2 , 2mM Spermine at pH 7.0, 100 counts/minute of 32 P-labelled RNA. The reactions were incubated at 37°C for 30 minutes then passed through a sandwich of filters (presoaked in matching buffer) in a vacuum manifold. Filters: size exclusion (Tuffryn) filter (Pall), Nitrocellulose filter (BioRad), Hybond-N+ charged nylon filter (GE BioSciences), and filter paper (Whatman). The filters were washed 3 times with wash buffer (25mM Tris HCl, 100mM KCl, 25mM MgCl 2 , pH 7.5) and allowed to dry for 3 hours. Reactions were quantified by phosphorimaging and data were fit using KaleidaGraph software.

Extended Data
Extended Data Figure 1

. Sequences and structures of RNAs
Top left: Sequence and secondary structure of the complete TYMV TLS and the Upstream Pseudoknot Domain (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 TLS 5,11 . Interestingly, the stop codon for the Coat protein is within the UPD (magenta). To the right and below: 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.  Fig. 2a). c, Crystal packing involving the anticodon 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, while not appearing to alter the overall anticodon 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 TLS's 3′ CCA (red, labeled) against an adjacent molecule (magenta) likely causes the CCA to adopt a foldedback conformation. 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 likely very different from that used by the TLSs that are histidylated or tyrosylated, which are very different in terms of their secondary structure and fold 6,9 . b, Same as panel a, but with the TLS modeled onto the bacterial homolog of eEF1A (EF-Tu) as in Extended Data Fig. 6. tRNA Phe 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. One crystal was used.     25 . c, The AC loop of the TLS (red) must swing into position to match tRNA's (cyan). d, TLS structure colored by relative crystallographic B-factor (high=red, blue=low).