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Template-dependent nucleotide addition in the reverse (3′-5′) direction by Thg1-like protein

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Science Advances  25 Mar 2016:
Vol. 2, no. 3, e1501397
DOI: 10.1126/sciadv.1501397
  • Fig. 1 Reaction schemes of 3′-5′ and 5′-3′ elongation.

    Top: Reaction scheme of 3′-5′ elongation by Thg1/TLP family proteins. Bottom: Reaction scheme of 5′-3′ elongation by DNA/RNA polymerases. In 3′-5′ elongation by Thg1/TLP family proteins, the 5′-monophosphate of the tRNA is first activated by ATP/GTP, followed by the actual elongation reaction. The energy of the incoming nucleotide is not used for its own addition but is reserved for the subsequent addition (head polymerization). In 5′-3′ elongation by DNA/RNA polymerases, the energy of the incoming nucleotide is used for its own addition (tail polymerization).

  • Fig. 2 Structure of the MaTLP complex with ppptRNAPheΔ1.

    Left: One molecule of the tRNA substrate (ppptRNAPheΔ1) is bound to the MaTLP dimer. The AB and CD dimers are further dimerized by the crystallographic twofold axis to form a tetrameric structure (dimer of dimers). Right: Left figure rotated by 90o. The CD dimer is omitted for clarity. The accepter stem of the tRNA is recognized by molecule A (yellow), and the elbow region by molecule B (blue). Residues important for binding are depicted in stick form. The β-hairpin region of molecule B is shown in red.

  • Fig. 3 Structural change of the tRNA (ppptRNAPheΔ1).

    Structural change of the tRNA (ppptRNAPheΔ1) accepter stem in MaTLP caused by insertion of GDPNP. (A) Structure before GDPNP binding. (B) Structure after GDPNP binding. (C) Superposition of the two structures showing movement of the 5′-end of the tRNA before (blue) and after (red) insertion of GDPNP. (D) Superposition of the 5′-end of the tRNA after GDPNP insertion (red) with GTP at the activation step (green), showing that both triphosphate moieties superpose well.

  • Fig. 4 Mutational analysis of the β-hairpin and anticodon binding region.

    The rates of guanylylation by various mutants were measured. Error bars represent the SD of three independent experiments. (A) Guanylylation of ppptRNAPheΔ1 and ppptRNAHisΔ1 by various TLP mutants. The activity using [α-32P]GTP, wild-type MaTLP, and ppptRNAPheΔ1 is denoted as 100. (B) Guanylylation of tRNAPheΔ1, tRNAPhe, and tRNAHisΔ−1 by various TLP mutants. The activity to tRNAPheΔ1 is about 10% of ppptRNAPheΔ1.

  • Fig. 5 Schematic representation of the 3′-5′ elongation mechanism.

    (A) The reaction center overlapped with two triphosphate binding sites. A, B, and C (in green) represent binding sites for Mg2+A, Mg2+B, and Mg2+C. P (in blue) represents the phosphate binding sites; O (in red) is the binding site for the deprotonated OH group. Important TLP residues for tRNA and Mg2+ binding are also shown. (B) Structure of the activation complex (corresponding to fig. S8). GTP/ATP binds to triphosphate binding site 1; the deprotonated OH group of the 5′-phosphate attacks the α-phosphate of GTP/ATP, and PPi (inorganic pyrophosphate) is released. (C) Possible structure after the activation step as suggested from the structure of (C′). (C′) Structure before the elongation reaction (corresponding to Fig. 3A). The 5′-triphosphate of the tRNA binds to the same site as for activation of the 5′-terminus of the tRNA in (B). (D) Structure of initiation of the elongation reaction (corresponding to Fig. 3B). The base of the incoming GTP forms a Watson-Crick hydrogen bond with the nucleotide at position 72 in the template chain and a base-stacking interaction with a neighboring base (G2). Movement of the 5′-terminal chain leaves the 5′-triphosphate of the tRNA in the same site as the activation step in (B). The 3′-OH of the incoming GTP is deprotonated by Mg2+A and attacks the α-phosphate to form a covalent bond. (E) After the elongation reaction, the triphosphate of the new nucleotide is placed on site 1, as in (C′), and is ready for the next reaction.

  • Fig. 6 Structures of template-dependent nucleotide elongation in the 3′-5′ and 5′-3′ directions.

    Symmetrical relationship between 3′-5′ elongation by TLP (this study) (left) and 5′-3′ elongation by T7 RNA polymerase [Protein Data Bank (PDB) ID: 1S76] (right). Red arrows represent elongation directions. In the 3′-5′ elongation reaction, the 3′-OH of the incoming nucleotide attacks the 5′-activated phosphate of the tRNA to form a phosphodiester bond, whereas in the 5′-3′ elongation reaction, the 3′-OH of the 3′-terminal nucleotide of the RNA attacks the activated phosphate of the incoming nucleotide to form a phosphodiester bond. Green spheres represent Mg2+ ions.

  • Table 1 Summary of data collection and refinement statistics.

    Values in parentheses are for the highest-resolution shell. PF, Photon Factory; Rmsd, root-mean-square deviation.

    MaTLP-apoMaTLP-GTPMaTLP-ppptRNAPheΔ1MaTLP-ppptRNAPheΔ1-GDPNP
    PDB ID5AXK5AXL5AXM5AXN
    Data collection
      BeamlineSPring-8 BL41XUSPring-8 BL41XUPF BL17APF BL5A
      Space groupC2221C2221I222I222
      Unit cell parameters a, b, c (Å)98.3, 120.5, 157.4103.1, 115.7, 144.975.3, 127.6, 143.882.3, 134.1, 147.4
      Wavelength (Å)0.97801.00000.973191.0000
      Resolution range (Å)50.0–2.29 (2.43–2.29)50.0–2.99 (3.17–2.99)50.0–2.21 (2.34–2.21)50.0–2.70 (2.87–2.70)
      Rmeas (%)*8.9 (76.3)15.2 (90.0)9.7 (74.4)11.0 (87.2)
      CC1/2 (%)99.8 (80.4)99.5 (81.2)99.9 (83.6)99.9 (83.5)
      〈I/σ(I)〉14.7 (2.8)12.0 (2.6)19.4 (3.2)16.9 (2.5)
      Completeness (%)98.3 (93.8)98.8 (93.4)99.7 (98.6)99.7 (99.3)
      Redundancy6.7 (6.6)7.2 (7.2)7.4 (7.3)8.1 (8.2)
    Refinement
      No. of reflections41,65017,58135,10222,669
      Rwork/Rfree (%)20.6/24.021.5/25.321.6/24.322.5/26.7
      No. of atoms
        Macromolecules3760362252475142
        Ligand/ion306836101
        Water89810216
      B-factors (Å2)
        Macromolecules57.068.445.357.3
        Ligand/ion60.586.246.659.9
        Water49.059.533.038.1
      Estimated coordinate error (Å)0.320.480.250.41
      Rmsd from ideal
        Bond lengths (Å)0.0090.0030.0030.003
        Bond angles (°)1.110.920.720.80

    *Rmeas = Σhkl {N(hkl)/[N(hkl) − 1]}1/2 Σi | Ii(hkl) − 〈I(hkl)〉 |/Σhkl Σi Ii(hkl), where 〈I(hkl)〉 and N(hkl) are the mean intensity of a set of equivalent reflections and the multiplicity, respectively.

    Rwork = Σhkl ||Fobs||Fcalc||hkl |Fobs|; Rfree was calculated for 5% randomly selected test sets that were not used in the refinement.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/3/e1501397/DC1

      Fig. S1. Structure of MaTLP-ppptRNAPheΔ1 with the final 2FoFc map.

      Fig. S2. Comparison of the tRNA binding structures of MaTLP and CaThg1.

      Fig. S3. Stereo drawings of the MaTLP active center.

      Fig. S4. Structure of the active center in the MaTLP-ppptRNAPheΔ1-GDPNP complex superposed with an omit map.

      Fig. S5. Multiple sequence alignment of Thg1/TLP enzymes.

      Fig. S6. tRNA binding experiments using gel filtration.

      Fig. S7. Binding models of TLP and tRNA truncation variants.

      Fig. S8. A model of the active center in the activation complex.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Structure of MaTLP-ppptRNAPheΔ1 with the final 2Fo Fc map.
      • Fig. S2. Comparison of the tRNA binding structures of MaTLP and CaThg1.
      • Fig. S3. Stereo drawings of the MaTLP active center.
      • Fig. S4. Structure of the active center in the MaTLP-ppptRNAPheΔ1-GDPNP complex superposed with an omit map.
      • Fig. S5. Multiple sequence alignment of Thg1/TLP enzymes.
      • Fig. S6. tRNA binding experiments using gel filtration.
      • Fig. S7. Binding models of TLP and tRNA truncation variants.
      • Fig. S8. A model of the active center in the activation complex.

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