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Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure

2003; Springer Nature; Volume: 22; Issue: 21 Linguagem: Inglês

10.1093/emboj/cdg563

1460-2075

Masaru Okabe, Kozo Tomita, Ryuichiro Ishitani, Ryohei Ishii, Nono Takeuchi, Fumio Arisaka, Osamu Nureki, Shigeyuki Yokoyama,

Genomics and Phylogenetic Studies

Article3 November 2003free access Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure Mayuko Okabe Mayuko Okabe Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Kozo Tomita Kozo Tomita Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba, 277-8562 Japan Search for more papers by this author Ryuichiro Ishitani Ryuichiro Ishitani Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Ryohei Ishii Ryohei Ishii Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Nono Takeuchi Nono Takeuchi Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba, 277-8562 Japan Search for more papers by this author Fumio Arisaka Fumio Arisaka Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501 Japan Search for more papers by this author Osamu Nureki Corresponding Author Osamu Nureki RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045 Japan PRESTO, JST, 4-1-8 Hon-cho, Kawaguchi-shi, Saitama, 332-0012 Japan Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501 Japan Search for more papers by this author Shigeyuki Yokoyama Shigeyuki Yokoyama Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045 Japan Search for more papers by this author Mayuko Okabe Mayuko Okabe Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Kozo Tomita Kozo Tomita Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba, 277-8562 Japan Search for more papers by this author Ryuichiro Ishitani Ryuichiro Ishitani Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Ryohei Ishii Ryohei Ishii Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan Search for more papers by this author Nono Takeuchi Nono Takeuchi Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba, 277-8562 Japan Search for more papers by this author Fumio Arisaka Fumio Arisaka Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501 Japan Search for more papers by this author Osamu Nureki Corresponding Author Osamu Nureki RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045 Japan PRESTO, JST, 4-1-8 Hon-cho, Kawaguchi-shi, Saitama, 332-0012 Japan Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501 Japan Search for more papers by this author Shigeyuki Yokoyama Shigeyuki Yokoyama Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045 Japan Search for more papers by this author Author Information Mayuko Okabe1, Kozo Tomita2, Ryuichiro Ishitani1, Ryohei Ishii1, Nono Takeuchi2, Fumio Arisaka5, Osamu Nureki 3,4,5 and Shigeyuki Yokoyama1,3 1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan 2Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba, 277-8562 Japan 3RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045 Japan 4PRESTO, JST, 4-1-8 Hon-cho, Kawaguchi-shi, Saitama, 332-0012 Japan 5Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, 226-8501 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5918-5927https://doi.org/10.1093/emboj/cdg563 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine–cytidine–adenosine' sequence onto the 3′ end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 Å resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes. Introduction The 3′-terminal CCA sequence (positions 74, 75 and 76) found in all mature tRNAs (Sprinzl and Cramer, 1979) is essential for various aspects of gene expression in all organisms. The CCA sequence is required for the aminoacylation by aminoacyl-tRNA synthetase, and for peptide-bond formation on ribosomes (Green and Noller, 1997; Nissen et al., 2000). The CCA sequence at the 3′ end of tRNAs is repaired and sometimes constructed de novo by a CCA-adding enzyme [ATP (CTP): tRNA nucleotidyltransferase] using CTP and ATP as substrates (Deutscher, 1982). The CCA-adding enzyme is present in all three primary kingdoms (Eubacteria, Eukarya and Archaea), indicating the conservation of its activity throughout evolution (Yue et al., 1996). The CCA-adding enzyme is indispensable in the many organisms in which some or all of the tRNA genes do not encode CCA (Aebi et al., 1990). Otherwise, the activity is advantageous for cell viability in the organisms, in which all of the tRNA genes encode CCA (Zhu and Deutscher, 1987). The CCA-adding enzyme is a member of the nucleotidyltransferase (NT) family which encompasses enzymes as diverse as poly(A) polymerase (PAP), terminal deoxynucleotidyltransferase (TdT), DNA polymerase β (pol β), glutamine synthase adenylyltransferase and kanamycin nucleotidyltransferase (KNT) (Holm and Sander, 1995; Martin and Keller, 1996; Yue et al., 1996). The CCA-adding enzyme is a remarkable enzyme among the NT family; it synthesizes the ordered CCA sequence at the 3′ end of the tRNA primer without the aid of a nucleic acid template. Furthermore, the enzyme strictly monitors the status of the tRNA 3′ end to reconstruct the complete CCA terminus. Based on biochemical and biophysical studies, several models have been proposed to explain the specific activity of this remarkable RNA polymerase (Deutscher, 1982; Shi et al., 1998a; Yue et al., 1998; Hou, 2000; Li et al., 2000, 2002). However, the details of the CCA addition mechanism have remained elusive for three decades. The CCA-adding enzymes are divided into two classes, the archaeal CCA-adding enzymes (class I) and the eubacterial/eukaryotic CCA-adding enzymes (class II), based on the sequence alignment (Yue et al., 1996). Although both classes of CCA-adding enzymes catalyze the same reaction in a defined fashion and cross-react with each other's tRNA substrates, no significant amino acid sequence similarity can be readily identified, except in the regions around the active site signature motif. Recently, the structures of the class II eubacterial and eukaryotic CCA-adding enzymes and/or their complexes with CTP and ATP were reported, revealing the nucleotide recognition mechanism (Li et al., 2002; Augustin et al., 2003). In contrast, the structure of a class I archaeal CCA-adding enzyme has not been solved, and the question remains as to how the class I CCA-adding enzyme recognizes CTP and ATP to catalyze the same reaction performed by the class II CCA-adding enzymes. In the present study, we have determined the crystal structures of the class I archaeal CCA-adding enzyme and its complexes with CTP and ATP. The structures provide significant implications for the mechanism and evolution of the remarkable CCA-adding activity. Results and discussion Structure determination of Archaeoglobus fulgidus CCA-adding enzyme The CCA-adding enzyme from the hyperthermophilic archaeon A.fulgidus (AfCCA) was overexpressed in Escherichia coli and crystallized. The apo structure was initially solved by multiwavelength anomalous dispersion (MAD) using the selenomethionine-labeled protein, and then the native structure was refined to an R factor of 19.8% (Rfree of 24.2%) using reflections up to 2.0 Å resolution. Subsequently, the model was refined against the datasets collected from crystals soaked in CTP or ATP. The structure of the CTP complex was refined to an R factor of 20.9% (Rfree of 24.9%) up to 2.0 Å resolution, while the structure of the ATP complex was refined to an R factor of 20.5% (Rfree of 26.0%) up to 2.7 Å resolution (Table I). Table 1. Summary of data collection and refinement statistics SeMet crystal Native +CTP +ATP Peak Edge Remote1 Remote2 Data collection Beam line BL41XU BL41XU BL41XU BL44XU Wavelength (Å) 0.9792 0.9795 0.9843 0.9753 0.9740 0.9704 1.0000 Resolution (Å) 50–3.0 50–3.0 50–3.0 50–3.0 50–2.0 50–2.0 50–2.7 Unique reflections 10 258 10 413 10 427 10 458 32 398 31 484 14 279 Redundancy 3.5 3.5 3.5 3.5 3.2 2.7 3.4 Completeness (%) 99.3 (98.5) 99.0 (96.7) 99.2 (97.7) 99.2 (97.9) 95.6 (87.3) 90.5 (80.3) 98.1 (80.7) I/σ(I) 20.1 (7.6) 20.6 (7.2) 20.4 (6.8) 19.9 (6.1) 20.0 (3.5) 12.8 (1.8) 13.8 (2.5) Rsym (%) 6.3 (12.0) 4.5 (12.1) 4.2 (11.6) 5.0 (13.5) 3.9 (16.8) 6.3 (35.6) 5.8 (21.3) Phasing statistics No. of sites 5 5 5 5 Phasing power Iso (acent/cent) 0.42/0.31 – 0.72/0.52 0.94/0.61 Rcullis Iso (acent/cent) 0.96/0.93 – 0.90/0.88 0.87/0.84 Ano 0.74 0.89 0.84 0.98 FOM (acent/cent) 0.47/0.41 Resolution (Å) 50–2.0 50–2.0 50–2.7 Reflections 32 398 31 484 14 279 Protein atoms 3560 3578 3578 Water oxygens 305 287 121 Rwork (%) 19.8 20.9 20.5 Rfree (%) 24.2 24.9 26.0 R.m.s.d. bond lengths (Å) 0.0061 0.0177 0.0065 R.m.s.d. bond angles (°) 1.27 1.70 1.48 R.m.s.d. dihedrals (°) 21.54 22.49 22.47 R.m.s.d. impropers (°) 0.89 1.16 1.02 0.47/0.41 Refinement statistics The numbers in parentheses are for the last shell. Rsym = Σ |Iavg − Ii|/ ΣIi Rcullis = Σ||FPH + FP| − FH(calc)|/Σ|FPH| Rwork = Σ|Fo − Fc|/ΣFo for reflections of work set. Rfree = Σ|Fo − Fc|/ΣFo for reflections of test set (10% of total reflections). Overall architecture of Archaeoglobus fulgidus CCA-adding enzyme The AfCCA protein structure consists of three main domains: the N-terminal, central and C-terminal domains. An additional tail domain is inserted into, but spatially sequestered from, the C-terminal domain. The overall structure of the class I AfCCA has a U-shape, with a large cleft (∼20 × 16 × 18 Å) surrounded by the three main domains (Figure 1A). The ATP or CTP molecule sits in the interdomain cleft between the N-terminal and central domains (Figure 1A). Strikingly, the overall architecture of AfCCA is fundamentally distinct from the 'sea-horse' architecture of the class II CCA-adding enzymes from Bacillus stearothermophilus (BstCCA) (Figure 1B) and human mitochondria (HmtCCA), and is rather similar to that of eukaryotic PAP (Figure 1B). In contrast with PAP, AfCCA exists as a dimer in the crystal, with the subunits related by crystallographic symmetry (Figure 1C). The inserted tail domain of one subunit closely interacts with the tail, central and C-terminal domains of the other subunit, resulting in a buried surface area of 2.5 × 103 Å2. Sedimentation equilibrium and velocity experiments revealed that AfCCA exists as a dimer in solution (Supplementary figure S1A available at The EMBO Journal Online) like the Sulfolobus shibatae CCA-adding enzyme (Li et al., 2000). In contrast, although there are two molecules in the crystal asymmetric unit, class II BstCCA and HmtCCA are likely to exist as monomers, since the buried surface area is small (512 Å2 in BstCCA) and the dimeric arrangements of BstCCA and HmtCCA are completely different. Therefore the oligomeric state is likely to differ between the class I and class II CCA-adding enzymes. An amino acid sequence alignment of the archaeal class I CCA-adding enzymes is shown in Figure 2. Figure 1.(A) Stereo view of the A.fulgidus CCA-adding enzyme structure. The N-terminal domain (residues 16–143), the central domain (residues 2–15 and 144–259), the C-terminal domain (residues 260–339 and 384–437) and the tail domain (residues 340–383) are shown in cyan, green, magenta and orange, respectively. The CTP molecule bound to the active site is colored red and the two bound metal ions are shown as yellow spheres. The α-helices and β-strands are designated by letters and numerals, respectively. (B) Comparison of the structures of bovine poly(A) polymerase complexed with 3′-dATP (left), class I A.fulgidus CCA-adding enzyme complexed with CTP (middle) and class II B.stearothermophilus CCA-adding enzyme complexed with ATP (right). The orientations of the catalytic domains are adjusted as in (A). The bound NTPs are colored red. (C) Two views of the A.fulgidus CCA-adding enzyme dimer. The two subunits are colored cyan and pink, with the respective tail domains shown in green and orange, respectively. CTPs and metal ions bound to the active sites are shown as in (A). Download figure Download PowerPoint Figure 2.Sequence alignment of the CCA-adding enzymes from five archaea (A.fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, Methanobacterium thermoautotrophicum and S.shibatae). The sequences of the N-terminal and central domains of bovine poly(A) polymerase were also aligned according to their structural composition by using O (Jones et al., 1991). Blue bold numbers indicate the number of residues not displayed. Important conserved residues are highlighted in red for catalytic carboxylates and in yellow for residues interacting with the nucleotides. Highly conserved residues are in red. The signature sequence that forms the helical-turn motif is surrounded by a blue box. The secondary structure elements of A.fulgidus CCA-adding enzyme are indicated above the alignment, and those of bovine poly(A) polymerase are indicated below the alignment, as chains (α-helices), narrow chains (310 helices), arrows (β-strands) or TT (turns). The four domains in A.fulgidus CCA-adding enzyme are color coded as in Figure 1A. The putative tRNA-binding regions are shown with an orange bar and a blue bar just above the alignment in the subunit that catalyzes the CCA-addition and the other subunit, respectively. Download figure Download PowerPoint The catalytic site of the nucleotidyltransfer reaction All NT family members, including pol β and other known polynucleotide polymerases, use the 'two-metal-ion catalytic' mechanism for the nucleotidyltransfer reaction (Brautigam and Steitz, 1998). In the structures of the known NTs, a metal ion(s) is coordinated in the active site (Figure 3) (Sakon et al., 1993; Pelletier et al., 1994; Bard et al., 2000; Martin et al., 2000). In the present structure of AfCCA, three carboxylates (Glu59, Asp61 and Asp110) are located in close proximity to each other and coordinate the catalytic Mg2+ ion(s) (Figures 3A and 4). In addition to Glu59 and Asp61, which were suggested to be catalytic carboxylates in the class I A.fulgidus CCA-adding enzyme (Asp53 and Asp55 in S.shibatae CCA-adding enzyme) (Yue et al., 1998), Asp110 (corresponding to Asp106 in S.shibatae CCA-adding enzyme) was now found to be the third carboxylate for the NT reaction (Figures 3A and 4). The mutation of Asp110 to Ala in the AfCCA enzyme resulted in a significant decrease in the rate of CMP and AMP incorporation into tRNA-DC (D is the discriminator nucleotide at position 73) and tRNA-DCC, respectively, in vitro (Figure 4D). In contrast, in the class II BstCCA, the third carboxylate has not yet been proved to be functionally important, and in the class II HmtCCA, it was not essential for catalysis (Augustin et al., 2003). In the CTP complex structure, the two Mg2+ ions interact with the three carboxylates in the active site (Figure 4A), while only one Mg2+ ion is bound to the active site in the ATP complex structure (Figure 4B). The three catalytic carboxylates of AfCCA are superimposable onto the corresponding three carboxylates of bovine PAP (Asp113, Asp115 and Asp167) (Figure 3B). The arrangements of the metal ions and the three carboxylates also correspond well to those observed in the structure of the pol β ternary complex (Figure 3D). Although the overall architectures of the class I and class II CCA-adding enzymes are different (Figure 1B), the class I N-terminal domain and the class II head domain have conserved amino acid residues and motifs. Between the two classes of CCA-adding enzymes, the five β-strands and two α-helices, which construct the catalytic core, can be superimposed with an r.m.s.d. of 2.1 Å for 62 corresponding Cα atoms. Moreover, the three carboxylates of BstCCA (Asp40, Asp42 and Glu79) are completely superimposable onto the three carboxylates of AfCCA (Glu59, Asp61 and Asp110) (Figure 3A and C). Therefore the catalytic cores of the class I and class II CCA-adding enzymes share a common ancestor, together with those of PAP and pol β, and catalyze the nucleotidyltransfer reaction by the same mechanism. Figure 3.Structural alignment of the conserved catalytic domains of (A) A.fulgidus CCA-adding enzyme (N-terminal domain), (B) bovine poly(A) polymerase (N-terminal domain), (C) B.stearothermophilus CCA-adding enzyme (Head domain) and (D) human pol β (Palm domain). The α-helices and β-strands are colored pink and blue, respectively. Three catalytic carboxylates are displayed. Metal ions are shown as yellow spheres. Download figure Download PowerPoint Figure 4.CTP and ATP molecules bound to the active site of A.fulgidus CCA-adding enzyme. Interactions between (A) CTP or (B) ATP and conserved amino acid residues of A.fulgidus CCA-adding enzyme are shown. The secondary structure elements are color coded as in Figure 1A. Hydrogen bonds are shown as dashed black lines. The superimposed simulated annealing omit map (gray) is contoured at 3.0σ. (C) Superimposition of CTP (green) and ATP (blue) bound to the active site of A.fulgidus CCA-adding enzyme. Three conserved carboxylates in the CTP-bound and ATP-bound forms are displayed with green and blue backbones, respectively. (D) The activities of CTP (left) and ATP (right) incorporation into tRNA-DC and tRNA-DCC, respectively, by A.fulgidus CCA-adding enzyme mutants. The bars on the graphs indicate the SDs of five independent experiments. Download figure Download PowerPoint Recognition of CTP and ATP Both CTP and ATP are bound at the same pocket in the AfCCA complex structures (Figure 4A and C), underlying the utilization of a single active site pocket for the CCA addition by the class I CCA-adding enzyme (Yue et al., 1998). This was also observed in the structure of the class II CCA-adding enzyme complexed with NTP (Li et al., 2002), despite the different overall architectures between the two classes (Figure 1B). The relative position of the CTP or ATP to the active site in AfCCA is similar to that in BstCCA (Figure 3C). A conserved residue in the helical turn motif located in the N-terminal domain, Ser47, contacts the γ-phosphate of ATP and CTP in AfCCA (Figure 4A and B). This interaction between the Ser47 residue and the γ-phosphate of the incoming nucleotide is commonly observed in the structures of PAP, pol β and KNT. The main-chain amide group of Leu54 provides a hydrogen bond with the γ-phosphate moiety of both CTP and ATP (Figure 4A and B). In addition, Ser47 and Leu54 from the N-terminal domain and Lys152 and Tyr161 from the central domain are also involved in the recognition of the γ- and β-phosphate moieties of both ATP and CTP (Figure 4A and B). These two amino acid residues are strictly conserved among the class I archaeal CCA-adding enzymes and PAPs (Figure 2). The base moieties of CTP and ATP are recognized by the same amino acid residues (Arg50, His133 and Tyr173) (Figure 4A and B). His133, conserved among the class I CCA-adding enzymes (Figure 2), forms a hydrogen bond with either the 6-NH2 group of adenine or the 4-NH2 group of cytosine. The base moieties of CTP and ATP each provide a stacking interaction with Tyr173, which is also conserved among the class I CCA-adding enzymes (Figure 2). In both the ATP and CTP complex structures, Asp218 forms hydrogen bonds with the Nϵ group of His133 and the OH group of Tyr173 (Figure 4A and B), suggesting that Asp218 stabilizes the spatial arrangement of the two amino acid residues involved in the binding of both CTP and ATP. Another amino acid residue, Arg50, forms a weak hydrogen bond (distance of 3.69 Å) with the O2 atom of cytosine, and is in close enough proximity (distance of 3.91 Å) to interact with the N3 atom of adenine (Figure 4A and B). Among these amino acids interacting with base moieties, only Arg50 is crucial for the in vitro incorporation of CMP and AMP into tRNA-DC and tRNA-DCC, respectively (Figure 4D), while the mutation of His133, Tyr173 or Asp218 affects neither CMP nor AMP incorporation in vitro at low nucleotide concentrations (Figure 4D). The smaller effect of the R50A mutation on AMP incorporation than on CMP incorporation is probably due to the weaker interaction of ATP with Arg50 than that of CTP with Arg50. The Ala mutations of Thr52 and Glu176, which interact with the β phosphate of ATP (Figure 4B), but not CTP, did not affect the NT activity (Figure 4D). In the class II BstCCA, the base moiety of ATP or CTP is commonly recognized by Arg157 and Glu154, located on the neck domain (Li et al., 2002). Although identical amino acids at a single active pocket recognize both ATP and CTP in class I AfCCA and class II BstCCA, the detailed mechanism of the nucleotide recognition significantly differs between the two enzyme families. First, the triphosphate moiety of ATP or CTP bound to class II BstCCA adopts a common extended conformation. The base moieties have the usual anti conformation. CTP bound to class I AfCCA adopts a similar extended triphosphate conformation, with the base moiety in an anti conformation (Figure 4A). In contrast, the ATP molecule bound to class I AfCCA adopts an unusual syn conformation, which is stabilized by a hydrogen bond between the α-phosphate group and the N3 atom of the adenine moiety (Figure 4B). As a result, the triphosphate moiety of ATP assumes a more stressed, kinked conformation, which may account for the exclusion of the second metal ion, as described above. These unusual conformations of the base and triphosphate moieties enable the 6-amino group and the N3 atom of adenine to be located at spatial positions quite similar to those of the 4-amino group and the O2 atom, respectively, of cytosine (only shifted by 1.01 Å and 0.25 Å, respectively), leading to the recognition of the functional groups by the common His133 and Arg50 residues (Figure 4C). Secondly, the nucleotide substrates are recognized by the N-terminal and central domains of class I AfCCA, while the nucleotides are recognized by only the neck domain of class II BstCCA. Thirdly, a comparison of the CTP-bound and ATP-bound structures of class I AfCCA reveals only slight conformational changes of the nucleotide-recognizing Arg50, His133, Tyr173, Ser47 and Tyr161 residues. This is probably due to the fact that the corresponding functional groups of CTP and ATP, which are recognized by AfCCA, are spatially located at almost the same position, as described above. In contrast, in the structures of the CTP-bound and ATP-bound class II BstCCAs, the 6-amino group and the N1 atom of adenine are shifted from the 4-amino group and the N3 atom of cytosine by 1.79 and 0.79 Å, respectively. Correspondingly, Arg157 and Asp154, which recognize the N1 (ATP) and N3 (CTP) atoms and the 6-amino (ATP) and 4-amino (CTP) groups, respectively, considerably change their conformations, thus exhibiting induced fit by nucleotide binding. On the other hand, as described above, the structures of the ATP and CTP triphosphate moieties significantly differ in AfCCA, such that the 3′OH group of the bound ATP excludes the second Mg2+ ion. In contrast, in the BstCCA structures, the triphosphate structures are almost the same between the ATP-bound and CTP-bound forms. As a result, in AfCCA, the carboxyl side chain of Asp61, which binds to the second Mg2+ ion and is directed toward the 3′OH of CTP (2.65 Å apart), rotates by about 90° to be 3.69 Å away from the 3′OH in the ATP-bound form. Therefore, in class I AfCCA, the 3′OH group of the bound CTP is likely to be more activated to attack nucleophilically the incoming NTP compared with the 3′OH group of the bound ATP, which may be related to the mechanism of the template-independent, but sequence-specific, addition of CCA by the enzyme. In class II BstCCA, this involvement of the active site metal was not identified. In the ATP-bound and CTP-bound forms of class II BstCCA, the 3′OH group of the NTP is more distant from the 'catalytic triad'; the shortest distance is 5.83 Å, between the 3′OH of CTP and the carboxyl group of Asp42. Therefore, in AfCCA, the bound NTP is located in the catalytically favorable position for the phosphoryl transfer reaction, as compared with the corresponding positions in BstCCA and the eukaryotic PAPs. Thus the detailed mechanisms not only of nucleotide recognition but also of the specific polymerization reaction are likely to differ between the two classes of CCA-adding enzymes. From the structures presented here, the mechanism of switching from CMP incorporation to AMP incorporation, so as to complete the CCA synthesis, is still elusive. A previous biochemical analysis revealed that S.shibatae CCA-adding enzyme still catalyzes CA addition to a tRNA UV crosslinked to the enzyme, and the same tRNA phosphate groups remain in contact with the enzyme during the incorporation of C and A (Shi et al., 1998a). These results indicate that the tRNA is fixed on the enzyme surface during the CCA-adding reaction, and the 3′-end refolds successively to enable C, C and A addition. Considering that a single amino acid (Arg50) is involved in both CMP and AMP incorporation into tRNAs in vitro (Figure 4D), it is likely that either the refolding nucleotide at the 3′ end of the tRNA or the ribonucleoprotein composed of the tRNA 3′ end and enzyme together (Shi et al., 1998a; Li et al., 2002) is involved in switching the specificity via the network of Arg50, His133 and Tyr173. The details of the switching mechanism will await the structure determination of the enzyme, tRNA and nucleotide complexes. tRNA-docking model The C-terminal domain of AfCCA consists of a four-stranded antiparallel β-sheet, flanked by one long and six short helices on both sides (Figure 1A). A portion of this domain is topologically similar to the RNA-recognition motif (RRM) of several RNA-binding proteins, such as ribosomal protein S6 [Z score of 5.1 using the Dali server (Holm and Sander, 1998)], and the splicing factor U2AF 35 kDa subunit (Z score of 5.1). The electrostatic surface potential of the dimeric form of AfCCA shows a highly biased distribution of charged residues (Figure 5). The back surface of the central domain and large portions of the C-terminal and tail domains are positively charged. The dimeric form adopts a V-shaped structure (Figure 1C), the inside of which is positively charged and topologically suitable for accomodating tRNAs (Figure 5A). Therefore a plausible tRNA-docking model was built in which one tRNA molecule is bound to the AfCCA dimer, considering that the top-half region of the tRNA body is recognized by the class I CCA-adding enzyme (Shi et al., 1998b) (Figure 5B and C). Such a 2:1 complex o