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-D
mini-DC
mini-DC
+ CTP
mini-DCC
P43212
P43212
P43212
P43212
Data collection
Space group
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)
Resolution (Å)
1.00
1.00
1.00
1.00
50 – 2.80
(2.64 – 2.55)*
50 – 2.70
(2.44 – 2.36)
50 – 2.50
(2.59 – 2.50)
50 – 2.80
(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)
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
103
169
163
110
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
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
0.45
0.40
0.42
0.55
Refinement
Solvent
Average B-factors
(Å2)
R.m.s. deviations
Coordinate error (Å)
*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
P43212
P43212
P43212
Data collection
Space group
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
Resolution (Å)
50 – 2.50
(2.59 – 2.50)*
50 – 2.77
(2.87 – 2.77)
50 – 2.60
(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)
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
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
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
Refinement
Average B-factors
(Å2)
R.m.s. deviations
*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.
Supplementary Movie 2: Close-up view of the CCA-adding dynamics in the catalytic
site.
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