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Structure of transfer RNAs: similarity and variability

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Abstract Transfer RNAs (tRNAs) are ancient molecules whose origin goes back to the beginning of life on Earth. Key partners in the ribosome‐translation machinery, tRNAs read genetic information on messenger RNA and deliver codon specified amino acids attached to their distal 3′‐extremity for peptide bond synthesis on the ribosome. In addition to this universal function, tRNAs participate in a wealth of other biological processes and undergo intricate maturation events. Our understanding of tRNA biology has been mainly phenomenological, but ongoing progress in structural biology is giving a robust physico‐chemical basis that explains many facets of tRNA functions. Advanced sequence analysis of tRNA genes and their RNA transcripts have uncovered rules that underly tRNA 2D folding and 3D L‐shaped architecture, as well as provided clues about their evolution. The increasing number of X‐ray structures of free, protein‐ and ribosome‐bound tRNA, reveal structural details accounting for the identity of the 22 tRNA families (one for each proteinogenic amino acid) and for the multifunctionality of a given family. Importantly, the structural role of post‐transcriptional tRNA modifications is being deciphered. On the other hand, the plasticity of tRNA structure during function has been illustrated using a variety of technical approaches that allow dynamical insights. The large range of structural properties not only allows tRNAs to be the key actors of translation, but also sustain a diversity of unrelated functions from which only a few have already been pinpointed. Many surprises can still be expected. WIREs RNA 2012, 3:37–61. doi: 10.1002/wrna.103 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Processing > tRNA Processing

The seminal structural foundations underlying the ongoing investigations on structure/function relationships in tRNAs. (a) 1964: Shape of bulk tRNA deduced from the displayed X‐ray small‐angle scattering data (note overlap of the theoretical scattering curve of the L‐shaped model with the experimental data9). (b) 1965: Nucleotide sequence (with modifications) of Sce tRNAAla folded in an elongated hairpin or a cloverleaf as published by Holley et al.10 (c) 1971: Generalized cloverleaf fold of tRNA (with standard numbering) established after sequencing two dozens tRNAs. Conserved and semiconserved residues are indicated in bold (R, purine; Y, pyrimidine; D, dihydrouridine; T, ribothymidine; Ψ, pseudouridine); the α‐ and β‐regions (2–4 and 2–6 nts, respectively) in D‐loops correspond to sequence stretches 5′‐ and 3′‐apart from conserved G18G19; extra‐sequences (first discovered in Sce tRNASer) are inserted between residues 47 and 48 and form the extra arm in the variable region. The modified nucleosides found in a selection of 10 tRNAs are given in standard abbreviations (note the systematic presence of D, T, and Ψ); *modified G and Ψ of unknown structure (details on modifications in http://modomics.genesilico.pl/). In 2011, the generalized cloverleaf remains almost unchanged for most cytoplasmic tRNAs1 but shows deviations for many mitochondrial tRNAs (mt‐tRNAs)28; in contrast, the number of modified nucleosides found in tRNAs increased from 23 to 92. (d) 1974: L‐shaped conformation of Sce tRNAPhe (computed with refined coordinates, 4tra).11,12 (e) 1980: Boomerang‐like L‐shape of Sce tRNAAsp (closed form, 2tra).13 The four constitutive helical stems of tRNA are colored in the 2D cloverleaf‐ and 3D‐foldings [yellow for the Amino acid Accepting Stem (AAS), orange for the T Stem Loop (TSL), green for the D Stem Loop (DSL) and blue for the Anticodon Stem Loop (ASL)]; the same abbreviations and colors for tRNA domains are used all along this essay. Three‐dimensional structures can be visualized in the user friendly Proteopedia free encyclopedia (http://www.proteopedia.org) for the 4‐digit PDB accession codes.

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tRNA dynamics. (a) Comparison of a same tRNA in different conformations, either free or associated with partners from the protein synthesis machinery, and superposition of two conformers under two orientations in the central part of the figure: (a1) closed (left, 2tra) and open form (right, 3tra) of free Sce tRNAAsp (to highlight opening of the D/T‐loop interaction); (a2) closed conformation of Sce tRNAAsp (left, 2tra) and the same tRNA (right, 1asy) in contact with Sce AspRS (to visualize conformational change of AC‐loop and closure of the angle between the two arms of the L); (a3) Eco tRNACys interacting with Eco CysRS (left, 1u0b) and Taq EF‐Tu (right, 1b23) (to highlight a movement of more than 10 Å of AC‐loop due to reorganization of the core consecutive to flipping out of U45 and formation of the new bt A10•U25•U45, Figure 5). Note local conformational changes occurring in loop and core regions and huge variations in the relative orientation of the two tRNA branches with angles varying from 65° to 110° and inter‐domain separations in the ∼57–66 Å and ∼58–80 Å range, for example, for P35–P56 and P35–P73 distances.3 (b) Putative signal transduction pathways from anticodon identity determinants to CCA end in Eco tRNAGln:GlnRS (1gtr) and Sce tRNAAsp:AspRS (1asy) complexes. Three‐dimensional‐structures can be visualized in the user friendly Proteopedia free encyclopedia (http://www.proteopedia.org) for the 4‐digit PDB accession codes.

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Structural elements sustaining L‐shaped architectures in the four groups of divergent mitochondrial tRNAs with atypical (a) and resected (b–d) cloverleaf folds. (a) Hsa mt‐tRNAAsp with atypical D‐ and T‐loops. (b) Cel mt‐tRNAAsp missing TSL. (c) Bta mt‐tRNASer(GCU) missing DSL. (d) Wha mt‐tRNAAla missing both TSL and DSL. A symmetry two‐fold axis highlights the AC‐ and AAS‐branches of the mt‐tRNAs in their 2D sequence representations. The core accounting for 3D‐folding (as defined for canonical tRNAs, see text) is given for the four tRNAs with the same color code as in Figure 5. The figure displays also the complete core with seven base layers in the experiment‐based model of Hsa mt‐tRNAAsp and shows how it simplifies from 7 to 1 layer upon resection of cloverleaf domains.

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tRNA on the Tth ribosome. (a) View of the 70S elongation complex at 3.1 Å resolution. The structure shows Eco tRNAPhe molecules in ribosomal A (Amino acid) site (pink) and P (Peptidyl‐transferase) and E (Exit) sites (purple) interacting with mRNA (with 5′ Shine‐Dalgarno sequence followed by a polyU tail). Components of 30S and 40S subunits are shown in transparent surface mode with 18S, 5S, and 23S rRNAs in orange, dark green, and light green, and proteins from the small and large subunits in yellow and light blue. The 3 interacting Eco tRNAPhe molecules are unmodified except at position 37 occupied by ms2i6A. (b) Close‐up view of codon:anticodon interactions in elongation complex shown in yellow, orange and olive green from 5′ to 3′ on mRNA. (c) Superposition of the tRNAPhe molecules occupying the E, P and A sites (backbones in yellow, orange, and olive green) seen in two orientations. PDB codes (3i8f) for 50S and (3i8g) for 30S with mRNA and tRNA.77 Three‐dimensional‐structures can be visualized in the user friendly Proteopedia free encyclopedia (http://www.proteopedia.org) for the 4‐digit PDB accession codes.

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Diversity in tertiary networks leading to L‐shaped 3D‐structures. (a) The standard networks (divided in a stacked core and a D/T‐loop interaction domain, see text) representative of a majority of class I and class II tRNAs as revealed in the structures of Sce tRNAAsp (2tra) and Tth tRNASer (with SerRS) (1ser) and their location in the tRNA 3D‐structures are displayed. A scheme recapitulates the organization of the core in seven base layers and its relationship with AAS, ASL and variable region (central part of the figure, with — for WC‐interaction, —· — for atypical interaction and X indicating the broken interactions in class II tRNAs). Bases participating in core interactions are colored blue, yellow, green, brown, light‐blue, magenta, and violet in layers 1–7. For Tth tRNASer note additional U20a (colored red in layer 1) for stabilization of the Levitt pair, shift of G9 (from layer 4) in replacement of G46 (in layer 3) and absence of A26 in layer 7 (not seen in crystal structure). Conserved residues responsible for the D/T‐loop interaction are colored in light blue (G18–U55), light‐grey (G19·C56) and orange (A57). Note that the 12 bp arrangement of the AC‐branch is continued by three base layers connecting D‐loop with T‐loop and variable region (R15·Y48, N59, and Y60) and in the AC‐loop (in the conformation for codon recognition) by an additional stacking of R37 and the three anticodon residues N36, N35 and N34, thus constituting a stack of 19 base layers. (b) Three atypical networks: Eco tRNACys (1b23, structure in the complex with EF‐Tu), Dha tRNAPyl (2zni) and Hsa tRNASec (3a3a). Tertiary networks (core and D/T‐loop interaction domain) and 3D‐structures are displayed in the same orientation as for canonical tRNAs and with the same color code, except for the D/T‐loop interaction domain of Dha tRNAPyl where the mimics of the 19–56 and 18·55 pairs are colored red (for U19–A56) and pink (for A18/G55 with G55 in syn conformation); conserved A57 is colored orange as in (a). Note in layer 1 the position of C59 in the T‐loop of tRNAPyl (colored red) that faces G14 (colored yellow), in layer 2 the G22–C13·G48 bt replacing A21/U8/A14 and in layer 7 G43a–C26b from the extended AC‐stem replacing N44–N26. Note also the D/T‐loop interactions in tRNASec that include G18·U55 and U16·U59 contacts and an unique U20·(G19–C56) bt. For rationalization, numbering of residues in all tRNAs discussed in this essay was based on that of class I tRNAs (Figure 2); this may introduce slight deviations from what given in PDB or original literature. Networks different from class I tRNAs can be easily distinguished when base layers become bicolor. Note that residues colored red in class II or in atypical tRNAs are out of the seminal tertiary network of class I tRNAs. Three‐dimensional‐structures can be visualized in the user friendly Proteopedia free encyclopedia (http://www.proteopedia.org) for the 4‐digit PDB accession codes.

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Diversity in tRNA primary and secondary structures. The 15 selected sequences (after intron removal and 5′‐ and 3′‐end maturation) are aligned according to secondary structural motifs (colored as in Figure 1) with anticodon residues in red and conserved and semiconserved nts in bold (sequences are without post‐transcriptional modifications but with CCA terminus not always encoded in the genes). (a) A first group of 10 canonical Eco and Sce tRNA sequences highlights overall sequence conservations and shows peculiarities in Eco tRNACys (U21, G48) and in tRNAs with long variable region (Eco tRNALeu(CAG) and Sce tRNASer(AGA)). Other peculiarities accounting for idiosyncratic features concern the length of the α, ß and variable regions (see text for details) notably in Sce tRNAAsp (α = 3, ß = 3, v = 4 nts) and Sce tRNAPhe (α = 4, ß = 2, v = 5 nts). (b) Structure‐based relationships between conserved and semiconserved nts that control 3D‐structure formation are indicated in interconnected framed boxes: for L‐shape formation via compact core (blue background), for T‐loop formation (orange background) and for the CCA tail and AC‐loop (white background). (c) A second group of five sequences illustrates mild to severe deviations (with sequence insertions as in Bta tRNASec(GCU) or deletions as in Dha tRNAPyl or in mt‐tRNAs where entire domains can be missing (e.g., DSL in Bta mt‐tRNASer(GCU) or TSL in Cel mt‐tRNAAsp). Structure‐based numbering in three nt‐long variable region of Dha tRNAPyl is C44, A46, and G48 (see text). (d) Secondary foldings deviating from canonical cloverleaf illustrated with the five deviating atypical sequences displayed above. Note the peculiarities in Hsa tRNASec (accepting stem of 9 bp instead of 7, D‐stem of 6 bp instead of 4, T‐stem of 4 bp instead of 5 in canonical tRNAs) and more generally the length variations in D‐ and T‐loops as well as the universal sequence conservation of AC‐loops and—NCCA 3′‐terminal tails.

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Crystallographic structures of tRNAs sustain common global shapes and conformational diversity. (a) Sce tRNAPhe (4tra), (b) Bta tRNALys3 (1fir), (c) Sce tRNAAsp in complex with AspRS (1asy), (d) Eco tRNAGln with GlnRS (1gtr), (e) Hsa tRNASec (3a3a), (f) Tth tRNASer (nts in AC‐loop and variable region not seen) with SerRS (1ser), (g) Dha tRNAPyl with PylRS (2zni), and (h) Pho tRNAVal with ArcTGT (1j2b). Proteins associated to tRNAs are represented as blue (green when dimeric) surfaces and secondary backbones. All tRNAs are shown in the same orientation. Note the similarity of the Sce tRNAPhe and Bta tRNALys3 structures (a and b) despite different post‐transcriptional modification patterns: mnm5s2U34, ms2t6A37 in Bta tRNALys3 and Cm32, Gm34, yW37, Ψ39 in Sce tRNAPhe; note also the sharp turn at U33 in AC‐loops when the ASL‐domain is not in interaction with a protein (a, b, e, g). Three‐dimensional‐structures can be visualized in the user friendly Proteopedia free encyclopedia (http://www.proteopedia.org) for the 4‐digit PDB accession codes.

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Post‐transcriptional modifications for tRNA structure formation. (a) Hyperediting in Ien mt‐tRNAPro showing the 18 C residues (circled) edited to U.41 (b) Base modification controlled cloverleaf folding in Hsa mt‐tRNALys. The methyl group of m1A9 shifts the equilibrium between the alternate hairpin and cloverleaf forms towards the cloverleaf fold in preventing formation of WC‐pairing of A9 with U64.48

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