1. Volume 26, Issue 2, Pages 189-203 (April 2007)
How U38, 39, and 40 of Many tRNAs Become the Targets for Pseudouridylation by TruA 
Sun Hur, Robert M. Stroud 
Molecular Cell 
Volume 26, Issue 2, Pages (April 2007)
DOI: /j.molcel
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2. Figure 1 Crystallographic Statistics
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Overview of the Structures of TruA•tRNA
(A) Superposition of the six complexes of TruA•tRNA by aligning the TruA Cα backbones. Each monomer of TruA consists of distinct N-terminal (yellow) and C-terminal (red) domains. tRNA (blue) binds to TruA across the dimerization interface.
(B) The ASL binds in the cleft formed between the N- and C-terminal domains, placing the nucleotides at 38–40 (in stick model) proximal to the catalytic Asp60 (cyan). The 15 nucleotide VAL, located between the ASL and the T stem loop, is disordered and could not be traced completely in most tRNAs.
(C) Surface representation of TruA•tRNAleu3i in two view angles shows that TruA interacts with the tRNA elbow, the D stem backbone, and the major/minor grooves of the ASL. Colors on the protein surface represent the electrostatic characteristics of the atoms: hydrophobic (white), positive (blue), negative (red), polar N (light blue), polar O (salmon), and sulfur (yellow). Colors on tRNA represent atom identities: C (green), O (red), P (orange), and N (blue).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 ASL-Out and ASL-In Conformations
(A) Stereo view. The superposition of the six tRNAs shows two distinct conformations of the ASL backbone (ASL-out and ASL-in) with different thermal factor profiles. The thermal factors are normalized relative to the average thermal factor of the bound protein Cα. ASL-out is more distant from the surface of TruA and exhibits significant flexibility, whereas ASL-in is bent toward TruA and shows thermal stability. A few nucleotides in ASL-out (typically nucleotides at positions 33–37) could not be traced in tRNAleu1, tRNAleu3iv, and tRNAleu3v.
(B) Spatial locations of target bases. (Left) Superposition of four ASL-out tRNAs with nucleotides at 38 (yellow), 39 (green), and 40 (blue) in stick model. (Right) Superposition of two ASL-in tRNAs with nucleotides 38, 39, and 40 similarly designated. In the insets, small spheres represent trajectories of the center of mass of the bases at 38–40 during the three 2 ns MD simulations. The starting structures (tRNAleu1 and tRNAleu3i) for the simulations are shown in stick model.
(C) Comparison of the surface of TruA in complex with ASL-in tRNAleu3ii (left) and ASL-out tRNAleu3i (right) (L1 in blue, L8 in magenta, D60 in red, R58 in orange, R110 in green, and Phe110 in cyan). Nucleotides at 38–40 are shown in stick model (only 40 is labeled). The cleft widths (double arrows) are measured between Cαs of R58 and Q170. Both proteins are in the same orientation, viewed from the bottom of the structures in (A).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Stacked and Flipped-Out Conformations of Target Bases
Difference maps (Fo − Fc, contoured at 2.5 σ) phased with the structure of (A) TruA•tRNAleu3ii (ASL-out), and (B) TruA•tRNAleu3iii (ASL-in), in which the nucleotides at 38–40 are omitted, show the target bases stacked in the ASL. Distances (dashed lines) measured between Asp60-Cγ and N1 in pyrimidines or N9 in purines indicate that the catalytic Asp60 cannot access the target bases in the stacked conformation in either ASL-out or ASL-in. (C) Stereo view of the active site of TruA•tRNAleu3i with TruB (PDB, 1R3E) active site superposed. The difference map (Fo − Fc) phased with the stacked conformation (green) shows the partial occupancy of G39 in the flipped-out conformation (cyan). The simulated annealing omit map is provided in Figure S2. Superposed TruB residues (yellow) align with homologous TruA residues. The cocrystallized reaction product, 5-fluoro-6-hydroxy Ψ (5f-6OH-Ψ) in the TruB structure aligns with the flipped-out G39. (D) Summary of the tRNA conformations.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
The Mechanism for Base-Flipping of U38, 39, and 40 from TMD Simulations
(A) TMD snapshots of TruA•tRNA when (a) U39 is stacked in the ASL, (b and c) U39 is partially flipped out, and (d) U39 is fully flipped out and positioned in the active site.
(B) The (Ad) structure viewed from a different angle to show the conformation of the ASL with the extruded U39.
(C) Pseudouridylation efficiencies of tRNAleu3, tRNAleu3/U39, and tRNAleu3/U40 by WT-TruA and R58A. Nucleotides mutated from tRNAleu3 in tRNAleu3/U39 and tRNAleu3/U40 are colored blue. Wobble base pair is indicated with ∗∗.
(D) Binding affinities of R58A to tRNAleu3, tRNAleu3/U39, or tRNAleu3/U40, compared with that of WT-TruA to tRNAleu3/U39. Quantitations of gel shift assays are plotted with ±standard deviation denoted by the error bars. Values of R2 for fitting are greater than 0.9 in all cases. The gel images are in Figure S3.
(E) The sequence alignment of TruA from bacteria and eukaryotes shows that Arg58 (green arrow) is strictly conserved in the TruA family. Below are the sequence alignments of selected enzymes from all five families of Ψ synthase, showing residues corresponding to residues 57–60 in E. coli TruA.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Modification Efficiencies of Variants of tRNAs
(A) tRNAleu3. The left-hand diagram of the ASL shows the target base in red; on the right, the introduced variations are colored blue. Initial velocity plots are color coded to correspond to the variant on the left. Modification efficiency is expressed as kcat/KM, s−1 μM−1.
(B) tRNAleu3/U40 and introduced variant, color coding as in (A).
(C) tRNAala1 variants and tRNAala1/tRNAleu3 chimeras. The top row shows tRNAala1 WT (blue cartoon) and tRNAleu3 (magenta cartoon). The adjacent diagrams show details of the ASLs and VALs. The red dotted line in the tRNAala1 ASL represents the additional base pairs predicted from the mfold program. The diagram of tRNAala1 shows introduced variations with modification efficiencies (kcat/KM, s−1 μM−1) adjacent to each variant. The bottom row shows tRNAala1/tRNAleu3 chimeras with modification efficiencies (kcat/KM, s−1 μM−1) below.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 The Proposed Model for the Target Site Recognition by TruA
In the initial docking complex (ASL-out), tRNA binds to TruA through the stable interactions at the elbow and the D stem while maintaining flexibility of the ASL. In the second step, the ASL bends toward TruA and forms intermediate states (ASL-in/base-stacked). Due to the dynamic nature of the ASL in the initial docking complex, three distinct intermediates can form, each placing a different target nucleotide adjacent to Arg58. Three distinct reactive conformations are formed when the nucleotide interacting with Arg58 flips out and becomes positioned at the active site near the catalytic Asp60.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 8 Comparison of RNA Recognition Mechanisms of TruA and TruB
Structural alignment of TruA and TruB using their Cα backbones (Guda et al., 2004). TruA (subunit A, orange; and subunit B, yellow) and TruB (PDB, 1R3E; core domain, slate; C-terminal domain, green; and thumb domain, cyan). The magnified view (lower left) shows the conservation of active site residues, including the catalytic Asp60 (bold italic). On the right, TruA and TruB are shown separately in the same orientation. The tRNA (magenta) bound to TruA is from the crystal structure (present work). The tRNA (magenta) bound to TruB is modeled based on the cocrystallized T stem loop (purple) and other RNA fragments (gray) that mimic the acceptor stem loop.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions