1. Structural Basis for the Major Role of O-Phosphoseryl-tRNA Kinase in the UGA-Specific Encoding of Selenocysteine 
Shiho Chiba, Yuzuru Itoh, Shun-ichi Sekine, Shigeyuki Yokoyama 
Molecular Cell 
Volume 39, Issue 3, Pages (August 2010)
DOI: /j.molcel
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2. Molecular Cell 2010 39, 410-420DOI: (10.1016/j.molcel.2010.07.018)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 The Crystal Structure of the tRNASec•PSTK Complex
(A) Orthogonal views of the overall structure of two M. kandleri tRNASec molecules in complex with an M. jannaschii PSTK dimer bound with an AMPPNP molecule (crystal type 1). The tRNASec molecules are colored light orange. The NTD, the CTD, and the linker of one subunit (chain A) of the PSTK homodimer are shown in blue, light blue, and black, respectively. The NTD and the CTD of the other subunit (chain B) are colored green and light green, respectively. The linker of chain B is disordered. The AMPPNP molecule is indicated by a space-filling model.
(B) tRNASec•PSTK interactions. The bases of the acceptor stem, AD linker, D arm, anticodon arm, extra arm, and T arm on tRNASec are colored pink, gray, light blue, green, yellow, and light orange, respectively. The discriminator base and the 3′-terminal CCA at positions 73–76 are colored purple. Amino acid residues on the NTD, the linker, and the CTD of PSTK are enclosed with green, black, and light green rectangles, respectively. See also Figure S1.
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4. Figure 2 Secondary Structures of tRNASec and tRNASer
(A and B) The cloverleaf models of tRNASec (A) and tRNASer (B) from M. kandleri.
(C and D) The cloverleaf models of tRNASec (C) and tRNASer (D) from M. jannaschii.
(E) The cloverleaf model of tRNASec from H. sapiens.
(F) The cloverleaf model of tRNASec from E. coli. The numbering is based on the previous report (Sturchler et al., 1993). Each arm of tRNASec is colored the same as Figure 1B. Archaeal/eukaryal tRNASec has a 9-bp acceptor stem and a 4-bp T stem (9/4 secondary structure configuration) (A, C, and E), and bacterial tRNASec has an 8-bp acceptor stem and a 5-bp T stem (8/5 configuration) (F), whereas the canonical tRNAs, including tRNASer, have a 7/5 configuration (B and D). See also Figure S2.
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5. Figure 3
Recognition of the D Stem and Outer Corner by the PSTK CTD for the Phosphorylation Activity
(A) Ribbon representation of the complex of M. kandleri tRNASec and M. jannaschii PSTK. Each arm of tRNASec is colored the same as in Figure 2A. Only one of the two tRNASec•PSTK complexes (chains A and C, complex II in crystal type 1) in the asymmetric unit is shown for clarity. See also Figure S1.
(B) The tRNASec outer corner. The base triple U20:G19:C56 and the amino acid residues interacting with G19 and U20, distributed in α8–α9, are indicated in stick models. Hydrogen bonds formed between bases, and those formed between a base and an amino acid residue are indicated by brown and black dashed lines, respectively.
(C) The D-loop cavity of tRNASec. G18, C20a, and U59 interact with Arg195 accommodated in the cavity.
(D) tRNASec phosphorylation activities of M. jannaschii PSTK. M. kandleri tRNASec, M. jannaschii FL-PSTK, and the NTD fragment were used for the assay.
(E) tRNASec phosphorylation activities of M. kandleri PSTK. M. kandleri tRNASec, FL-PSTK, and the NTD fragment were used. The error bars indicate SEM in (D) and (E). Each experiment was performed 4–6 times, and the values were averaged.
(F) Binding affinity of PSTK with M. kandleri tRNASec. Each reaction (3.0 μl) contained 25 μM tRNASec (See Experimental Procedures). Free tRNASec and tRNASec complexed with the FL-PSTK or the CTD fragment were resolved by native PAGE (7.0% acrylamide). tRNA was detected by toluidine blue staining. See also Figure S3.
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6. Figure 4
The Recognition of the Acceptor Stem, the Discriminator Base, and the CCA End of tRNASec
(A) The interactions between the acceptor stem (pink) and the PSTK NTD (green) in complex I. The phosphate backbone of the tRNASec acceptor stem hydrogen bonds with the main chains and/or the side chains of Arg84, Lys140, and Trp141 of the NTD. See also Figure S1.
(B) The base-specific acceptor-stem•NTD interaction. Hydrogen bonding interactions between G2:C71 or C3:G70 and Lys138 or Tyr139 are indicated.
(C) Two tRNASec molecules are superposed, based on the phosphorus atoms of the T arm (yellow). The tRNASec acceptor stems with and without the interaction with Lys138 and Tyr139 (chains D and C in complexes I and II, respectively, from crystal type 1) are shown in pink and magenta, respectively. The distances between the phosphorus atoms of G1 and C67 on chains C and D are about 11 and 19 Å, respectively. The major groove of the acceptor stem is enlarged when the hydrogen bonds with Lys138 and Tyr139 are formed.
(D) tRNASec phosphorylation activities of M. jannaschii PSTK against M. kandleri tRNASec. M. jannaschii WT PSTK, K138A-Y139A, and the K138A-Y139A-mutated NTD fragment were used for the assay. The error bars indicate SEM. Each experiment was performed 4–6 times, and the values were averaged.
(E) The interactions of the discriminator base G73 with PSTK. Tyr79 stacks with G73 and hydrogen bonds with the 5′ phosphate group of C74.
(F) The view of G73 orthogonal to (E). G73 interacts with the side chain and the main chain NH of Ser81 and the side chain of Asp133.
(G) The interactions between C74 and PSTK. The base of C74 hydrogen bonds with Lys55 and Ser81 and interacts with the side chain of Met82.
(H) The interactions between C75-A76 and PSTK. C75 and A76 fit into the cleft formed by hydrophobic residues. The ribose moiety of C75 stacks with the salt-bridged Arg40 and Glu51. See also Figures S1 and S4.
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7. Figure 5 tRNASer•PSTK Docking Model
(A) Comparison of the cloverleaf models of M. kandleri tRNASec and T. thermophilus tRNASer.
(B) T. thermophilus tRNASer (PDB ID: 1SER) superposed on M. kandleri tRNASec in the tRNASec•PSTK complex structure (complex II) based on the phosphorus atoms in their T arms. The D and T arms of tRNASec and those of tRNASer are shown in light blue, light orange, blue, and orange, respectively. The PSTK CTD is shown in both ribbon and surface models (green). The tRNASer D stem is not close enough to interact with the CTD, and the D-loop bases clash with the CTD.
(C) tRNASer•CTD docking model based on the T arm, from the same viewpoint as in (B). The CTD atoms clashing with the tRNASer D loop are colored red. Those interacting with tRNASec, but not close enough to interact with tRNASer, are colored cyan.
(D) tRNASer superposed on tRNASec in the tRNASec•PSTK complex structure, based on the phosphorus atoms in their D stems. The D loop of tRNASer severely clashes with the CTD.
(E) tRNASer•CTD docking model based on the D stem, from the same viewpoint as in (D). The CTD atoms clashing with the tRNASer D loop are colored red. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Proposed Scheme of tRNASec Transportation
PSTK binds Ser-tRNASec (1) and phosphorylates the seryl moiety of Ser-tRNASec (2). The NTD of PSTK dissociates from the produced phosphoseryl moiety of Sep-tRNASec, and in exchange, SepSecS associates with Sep-tRNASec, with no steric hindrance with the CTD of PSTK (3). SepSecS replaces the phosphoseryl moiety of Sep-tRNASec with the selenocysteinyl moiety, using selenophosphate (4). SepSecS dissociates from Sec-tRNASec, and the NTD of PSTK then rebinds and protects the selenocysteinyl moiety (5). While the PSTK NTD dissociates from the selenocysteinyl moiety of Sep-tRNASec, EF-Sec associates with Sep-tRNASec, with no steric hindrance with the CTD of PSTK (6). Finally, PSTK dissociates from the Sec-tRNASec•EF-Sec complex (7).
Molecular Cell  , DOI: ( /j.molcel )
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