Supplementary Information s35

SUPPLEMENTARY INFORMATION For Tomita et al.

Supplementary Discussion:

Crystal improvement by sulfate ion:

The inclusion of (NH4)2SO4 in the crystallization solution improved the quality of the crystals. While the recent complex crystal structures of AfCCA and RNA duplex mimics of the tRNA acceptor stem suggest that the 3'-terminal C74 or C75 is disordered in the absence of an incoming nucleotide14, the electron densities of the 3'-terminal C74 and C75 are clearly visible in our AfCCA binary mini-DC and mini-DCC stage complex structures, respectively. In both of the present mini-DC and mini-DCC stage structures, the electron density of a sulfate ion is visible in a position corresponding to that of the - phosphate of an incoming nucleotide in the mini-DC+CTP and mini-DCC+ATP stages (Supplementary Fig. S3). The presence of a sulfate ion in the present structures, but not in the previous structures, is due to differences in the crystallization conditions. The sulfate ion hydrogen-bonds to Lys152, Tyr161 and Ser47. It is reasonable to assume that the sulfate binding site corresponds to that of the phosphate ion of the pyrophosphate (PPi) produced by nucleotidyl transfer reaction. Therefore, our mini-DC and mini-DC stage structures may represent stages just after the nucleotidyl transfer reaction, and PPi remains on the enzyme. The tight binding of the sulfate ion might explain the improved quality of our binary crystals.

Mini-D+CTP stage: For the structure of the mini-D+CTP stage, we soaked CTP into the mini-D stage crystals. However, we could not observe an electron density for the incoming CTP. Co- crystallization of AfCCA, tRNAmini-D73 and CTP under similar conditions was not successful. The structures of the mini-D and mini-DC stages display the “open” conformation, and the recognition of the primer RNAs by the catalytic sites of both the mini-D and mini-DC stages is almost the same, except for the “+1” shift of the primer RNA (Fig. 3, a and b). Therefore, it is reasonable to propose that a similar “open” to “closed” conformational transition is triggered by CTP accommodation in the mini- D+CTP stage, with A73 flipping for CMP acceptance, where the CTP ligand is similarly selected by “knock-in” dynamics. Moreover, recent biochemical experiments and molecular modeling suggested that A73 is flipped and the 3'-OH group is located in the proximity of the catalytic triad in the mini-D+CTP stage24. After the first C-addition at position 74, both the 3'- and 5'-termini of the tRNA primer would then track back to accept the second CMP (Fig. 3b).

Catalytic Mg2+ ion: In the structures of mini-DC+CTP and mini-DCC+ATP stages, we could not observe a clear electron density for a catalytic magnesium, which would activate the nucleophilicity of the primer 3'-OH, although we did observe a Mg2+ ion beside the -, - and -phosphate groups of the NTP. We verified divalent cation binding by soaking in manganese ions (Supplementary Fig. S6), leading to the absence of the second catalytic Mg2+ ion near the catalytic triad and the primer 3-OH group. This may be due to the presence of 0.2 M lithium citrate, which might have chelated the Mg2+ ion. This lack of a catalytic Mg2+ ion may prevent CMP or AMP addition from proceeding. These data indicate that these stages represent a point just preceding the nucleotidyl transfer reaction.

Does the class-II CCA-adding enzyme employ the same knock-in-and-lock dynamics ?: The CCA-adding enzymes are divided into two classes: archaeal CCA-adding enzyme (class-I), and eubacterial and eukaryotic CCA-adding enzymes and eubacterial CC- and A-adding enzymes (class-II)9,25,26. The Class-I and class-II enzymes share no structural homology except for the N-terminal polymerase domain conserved in all nucleotidyltransferase enzymes15,16,27. The class I CCA-adding enzyme shares structural homology with the eukaryotic (class-I) poly-A polymerase (PAP)15,16,28,29. The class-I PAP has a shorter -turn motif28,29, and lacks the tail domain anchoring the RNA primer, which might allow the progressive “open” to “closed” conformational transition and the primer translocation towards the continuous polyadenylation reaction. It was recently reported that a chimeric protein, consisting of the NH2-terminal domain of E. coli PAP and the COOH-terminal domain of E. coli class-II CCA-adding enzyme, displays CCA- adding activity in vitro30. This suggests that the nucleotide-selecting element might reside outside the catalytic domain in the class II CCA-adding enzymes. Since the overall architectures of the class I and class II CCA-adding enzymes are basically different15,16,27, it remains unclear whether the class I and class II CCA-adding enzymes employ the same mechanisms of nucleotide selection and polymerization dynamics10,14- 16,27,31. The recently reported crystal structure of the class-II CCA-adding enzyme, in a complex with the A-lacking tRNA primer and ATP, revealed that ATP selection occurs in a catalytically inactive “open” conformation (pre-insertion state)10, as in the conventional RNA polymerases2,3. Nevertheless, the class-II CCA-adding enzyme enfolds the tRNA acceptor-TC helix and recognizes CTP and ATP using identical amino acid residues10,27, as in the class-I CCA-adding enzyme14-16, and the structural changes of the enzyme were suggested to discriminate between CTP and ATP at a given addition step10,27. Therefore, the present “knock-in-and-lock” polymerization dynamics, ensured by the anchoring of the tRNA TC-loop, might be a common mechanism underlying the progressive and specific CCA-addition by the class I and class II CCA- adding enzymes, although the class II CCA-adding enzyme employs a more enzyme- assisted nucleotide selection mechanism10,27.

Telomerase-like function of CCA-adding enzymes The CCA-sequence is found at the 3'-terminus of the tRNA-like structures of many plant virus RNAs, and the CCA is required for the replication of the RNA genome32. The 3'-terminal tRNA-like structures of several plant virus RNAs reportedly interact with CCA-adding enzyme, and the 3'-terminal CCA is repaired by class II CCA-adding enzymes (from E. coli and yeast) in vitro33 and probably in vivo by host class II CCA- adding enzymes34. These observations imply that the 3'-terminal CCA sequence and the CCA-adding enzyme act like a telomere and a telomerase, respectively35,36. The three- dimensional folding of the 3'-terminal region of plant viral RNAs, which contain the pseudo knot motif, is reportedly quite similar to that of the acceptor-TC helix of canonical tRNA37. Considering the similar RNA primer recognition mechanism between the class I and class II CCA-adding enzymes10,14, the class I CCA-adding enzyme also might be able to add CCA to the 3'-terminus of the tRNA-like structures of plant virus RNAs in vitro. Our preliminary docking analysis suggests that pseudo knot RNA (derived from PDB: 1HVU) possibly binds the present class-I CCA-adding enzyme (data not shown).

Supplementary Methods:

Crystallization and data collection We co-crystallized CCA-adding enzyme from Archaeoglobus fulgidus (AfCCA) with four distinct tRNA mini-helices with CCA-lacking, CA-lacking, A-lacking and mature termini, and in the presence or absence of an appropriate incoming nucleotide. These mini-helices act as efficiently as full-length tRNA substrates; therefore, the corresponding structures reflect the natural reaction stages during CCA-addition. All the binary and ternary complex crystals belong to the space group P43212, and contain one complex molecule in the asymmetric unit. For the crystallization of AfCCA binary complex with RNA, equal molar amounts of enzyme (110 M each) and RNA were mixed. One l of protein/RNA solution was mixed with one l of crystallization solution, containing 50 mM HEPES (pH 7.5), 80 mM (NH4)2SO4, 0.2 M tri-lithium citrate and 20% (v/v) PEG4000, and the drop solution was equilibrated against the reservoir solution at 20°C by the hanging drop vapor diffusion method. After one month, plate-like complex crystals were obtained. In the absence of (NH4)2SO4, the crystals were quite thin, and the quality of the diffraction data was not sufficient for structure determination, as described in the Supplementary discussion. For the preparation of the ternary complex with an incoming NTP, the crystals were soaked in a reservoir solution containing 3 mM NTP (ATP or CTP) at 20°C for 3 hours. The crystals were cryo-protected with 20% (v/v) ethylene glycol and were flash-frozen in a 100-K nitrogen stream, and the data were collected at the beam-line BL41XU of SPring-8 (Harima, Japan) and the beam-line NW-12 or BL5 of KEK (Tsukuba, Japan). Intriguingly, the “open” to “closed” conformational transition, may affect the differences in the cell dimensions of the crystals: the c-axis for the “open” structures is more than 10 Å longer than that for the “closed” structures (Supplementary Table S1) Supplementary Table S1 Data collection and refinement statistics for mini-D, mini-DC, mini-DC+CTP, and mini-DCC stage structures. mini-DC mini-D mini-DC mini-DCC + CTP Data collection

Space group P43212 P43212 P43212 P43212 Cell dimensions a (= b) (Å) 58.1 58.0 58.0 57.9 c (Å) 441.9 441.7 429.5 429.3 Wavelength (Å) 1.00 1.00 1.00 1.00 Resolution (Å) 50 – 2.80 50 – 2.70 50 – 2.50 50 – 2.80 (2.64 – 2.55)* (2.44 – 2.36) (2.59 – 2.50) (2.90 – 2.80)

Rsym 0.124 (0.259) 0.117 (0.360) 0.097 (0.255) 0.132 (0.256) I/ (I) 26.6 (3.58) 40.3 (7.04) 33.9 (4.47) 11.7 (1.53) Completeness (%) 99.0 (97.7) 99.8 (100) 97.8 (95.4) 90.9 (60.6) Redundancy 9.1 (5.6) 13.1 (12.6) 8.3 (5.7) 6.9 (2.2) Refinement Resolution (Å) 2.80 2.70 2.50 2.80 No. reflections 19376 21573 25932 17014

Rwork/ Rfree 0.236/0.293 0.219/0.273 0.224/0.257 0.208/0.281 No. atoms 4427 4513 4532 4474 Protein 3630 3630 3630 3630 RNA 684 704 704 724 Nucleotide — — 29 — Ion 10 10 6 10 Solvent 103 169 163 110 Average B-factors (Å2) 56.8 54.6 47.3 39.2 Protein 51.8 50.0 45.2 37.7 RNA 82.8 76.4 58.2 47.1 Nucleotide — — 43.7 — Ion 85.6 82.3 76.1 70.3 Solvent 57.9 59.9 47.3 31.6 R.m.s. deviations Bond lengths (Å) 0.008 0.007 0.007 0.007 Bond angles (˚) 1.3 1.2 1.2 1.2 Dihedral angles (˚) 21.0 20.8 21.2 20.9 Improper angles (˚) 1.07 1.05 1.03 1.00 Coordinate error (Å) 0.45 0.40 0.42 0.55 *Highest resolution shell is shown in parentheses. Supplementary Table S1 (continued) Data collection and refinement statistics for mini-DCC+ATP, mini-DCCA, and mini-DCC+CTP stage structures.

mini-DCC+ ATP mini-DCCA mini-DCC+ CTP Data collection

Space group P43212 P43212 P43212 Cell dimensions a (= b) (Å) 58.2 58.0 58.004 c (Å) 427.5 428.5 431.498 Wavelength (Å) 1.00 1.00 1.00 50 – 2.50 50 – 2.77 50 – 2.60 Resolution (Å) (2.59 – 2.50)* (2.87 – 2.77) (2.64 – 2.60) Rsym 0.083 (0.223) 0.138 (0.383) 0.131 (0.363) I/ (I) 37.9 (8.77) 10.6 (1.99) 29.6 (5.13) Completeness (%) 97.7 (95.1) 96.9 (94.8) 96.1 (95.8) Redundancy 5.9 (5.2) 6.5 (3.6) 7.5 (5.7) Refinement Resolution (Å) 2.50 2.80 2.6 No. reflections 25956 18344 22537

Rwork/Rfree 0.213/0.261 0.215/0.282 0.207/0.253 No. atoms 4575 4498 4512 Protein 3630 3630 3579 RNA 724 746 724 Nucleotide 31 — 29 Ion 6 10 6 Solvent 184 112 174 Average B-factors (Å2) 32.3 35.5 45.3 Protein 31.0 33.9 43.2 RNA 38.6 43.7 56.6 Nucleotide 20.3 — 40.37 Ion 59.1 77.2 74.7 Solvent 35.4 31.5 42.8 R.m.s. deviations Bond lengths (Å) 0.007 0.007 0.006 Bond angles (˚) 1.2 1.2 1.2 Dihedral angles (˚) 20.8 20.7 20.4 Improper angles (˚) 1.00 0.98 1.00 Coordinate error (Å) 0.35 0.45 0.36 *Highest resolution shell is shown in parentheses.

Supplementary Movie Legends:

Supplementary Movie 1: CCA-adding dynamics of the CCA-adding enzyme, primer tRNA and incoming NTP, highlighting the “open” to “closed” conformational transition of the enzyme.

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