supporting information - pnas · l453 r332 l334 g341 t339 f438 h338 r435 n431 d425 a276 l461 p418...
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Supporting InformationHamill et al. 10.1073/pnas.1003505107SI TextSI Methods. RNA preparation. For shorter RNAs, oligomers werepurchased from Dharmacon and gel purified. Duplexes, basedon the acceptor stem of tRNAAla, were obtained by annealing3′-CUCGCCGCGA-5′ and 50-GAGCGCGCUðAÞn-30. A 5′end-labeled strand was mixed with a 1.2-fold excess of the com-plimentary strand, heated to 95 °C, and slowly cooled to roomtemperature. The duplexes were purified by 15% native PAGE.
The tRNAs were in vitro transcribed from DNA templatesgenerated by PCR using overlapping primers. The DNA templateincluded the T7 promoter sequence. Sequences for the tRNAscorresponded to the wild-type and mutant tRNAAla (describedin ref. 1) and tRNAPhe (Fig. 4A). RNA transcripts were 5′end-labeled, gel purified, and resuspended in refolding buffer(5 mM MgCl2, 50 mM NaCl, and 20 mM Tris, pH 7.6). ThetRNAs were denatured by heating to 65 °C for 1 min, thenincubated for ∼2 min either on ice (tRNAAla) or at room tem-perature (tRNAPhe).
Protein constructs. The DNA sequence corresponding to residues161–481 of Trf4p was cloned into the pGEX-4T3 vector forexpression in Escherichia coli. The resulting construct has anN-terminal GST-tag cleavable by thrombin protease. The con-struct for the Trf4p D293A mutant that was used in crystallizationwas generated by site-directed mutagenesis from the wild-typeconstruct (Quickchange system, Stratagene).
The coding sequences for truncated versions of Air2p consist-ing of either residues 58–198 or 118–198 were cloned into thepCDF vector (Novagen). Each construct has an N-terminalhexahistidine tag that is cleavable by thrombin protease. Con-structs for zinc knuckle mutants were made by site-directedmutagenesis of the plasmid coding for Air2p residues 58–198.In these mutants, individual CX2CX4-5HX4C sequences in thezinc knuckle motifs were replaced with a hexaserine linker.
Protein expression and purification.E. coli BL21 cells were cotrans-formed with plasmids containing genes for Trf4p and Air2p. Cellswere grown at 37 °C in LB medium supplemented with 100 mMzinc until they reached an OD600 between 0.6 and 0.8. They werethen shifted to 20 °C, and protein production was induced with0.5 mM IPTG. Cells were harvested 18 h after induction.
The Trf4p/Air2p heterodimer was isolated by Ni-NTA chroma-tography (Qiagen) and bound to GST Sepharose (GE Health-care). Thrombin protease (350U per 4 L of cell culture) wasadded to cleave the hexahistidine and the GST tags from Air2pand Trf4p, respectively, and to elute Trf4p/Air2p from the GST-resin. Overnight treatment at 4 °C was sufficient for 100%cleavage. Eluted protein was run over a Mono-Q 500 column(GE Healthcare) equilibrated with 75 mM NaCl, 5 mM MgCl2,50 mM Tris, pH 7.6, and 5% glycerol (vol∕vol), and collected asthe flow through. Finally, the complex was purified by gel filtrationon a Superdex-200 column in buffer containing 15mMTris pH7.6,150 mM NaCl, and 5 mM MgCl2. The buffer for protein used inpolyadenylation assays was supplemented with 12% glycerol, andthe protein was aliquoted, then flash-frozen, and stored at −80 °Cuntil use.
Protein quantitation for polyadenylation assays. Protein concentra-tions were initially estimated from optical density measurements(280 nm), followed by quantitation on SDS-polyacrylamidegels. In comparing Trf4p∕Air2pZK1-5, mutant versions of Trf4p∕Air2pZK1-5, and Trf4p∕Air2pZK4-5 constructs, concentrationswere judged similar if the intensity of the Trf4p band on theSDS-PAGE gel was similar. Additionally, at these concentrations,the constructs exhibited similar activity on short A5 oligonucleo-tides (Fig. 1E).
1. Vanacova S et al. (2005) A new yeast poly(A) polymerase complex involved in RNAquality control. PLoS Biol 3:e189.
GST-Trf4p
His6-Air2p
MW 1 2 3
Fig. S1. Coomassie stained gel of Trf4p/Air2p complexes. Lanes 1 and 2 show purified Trf4p/Air2p complexes, where the N and C termini were removed. Lane 1shows the complex with two zinc knuckle modules, Trf4p(161-481)/Air2p(119-198), and lane 2 shows the complex with five zinc knuckles, Trf4p(161–481)/Air2p(58–198). These complexes were used in polyadenylation assays. Lane 3 shows a complex of GST-Trf4p∕His6-Air2p containing the full-length proteins. Theproteins were coexpressed and purified using Ni-NTA resin and GST Sepharose. Arrows indicate the full-length proteins. Both full-length proteins were severelydegraded during purification.
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pGCGGAUU
CGCUUAA
CCA
AOH
CCAGA
GGUCU
G
C
A
U
C
G
A
U
C
G
G
U
A
C
G
G
C
AU
UG
G
C
A
C UAG
CU
GAA
UC
AGGGU
U G A
1
20
35
55
70
C
G
G
A
U
C U G U G
C U C GGGUCU
o(T-stem)
(D-stem)
(anticodon stem)
[5ZK protein]
BA
lane 1 3 5 7 9 11 13 15
WT D-stem anticodon T -stem stem
Fig. S2. The Trf4p∕Air2pZK1-5 complex polyadenylates tRNAPhe mutants more rapidly than the wild-type tRNA. (A) Schematic of tRNAPhe mutants used inpolyadenylation assay in B. The mutations designed to disrupt secondary structure of the D-, anticodon, or T-stem are shown in black boxes. (B) Representativegel showing extent of polyadenylation for wild-type (lanes 1–4) and mutant tRNAPhe substrates. End-labeled tRNAPhe (25 fmol) was reacted with varyingamounts of a Trf4p/Air2p complex containing all five zinc knuckles (no protein in lanes 1, 5, 9, and 13; 0.5 pmol in lanes 2, 6, 10, and 14; 1.0 pmol in lanes3, 7, 11, and 15; or 2 pmol in lanes 4, 8, 12, and 16).
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Fig. S3. Alignment of the S. cerevisiae Trf4p sequence with sequences of Trf4/Trf5 family proteins in Saccharomyces cerevisiae (TRF5), Schizosaccharomycespombe (CID14), humans (PAPD5), Gallus gallus (LOC770425), Xenopus laevis (LOC445836), Drosophila melanogaster (TRF4-1), and Caenorhabditis elegans(GLD4). Residues that are highly or well conserved are highlighted in blue and cyan, respectively, and are mapped onto the Trf4p surface in Fig. 3 C–E.The three aspartates in the polymerase catalytic triad are orange, and the nucleotide recognition sequence of Trf4p is boxed. Secondary structure elementsin Trf4p (see Fig. 2) are indicated by bars.
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C
D
E
F459N456
T455
I175S176
V374I377
D177
N371I366
E378
D367
D365R360
T363 P359H358 E381
N386K444
R441
M357
Y403V402
F404
G388V392
L382
F354
D391D390
L394I308
B
V146
I144
Y143
A142
R141W140
R135
I139
L198
N197F193 S191
N197V185
R184S183
R181
Y165
Y163
H160
A
E196
R200P192
P233S235
D236/D238/D293K282
V272
V270
S473E182
I474
I478L453
R332
L334G341
T339
F438H338
R435N431D425A276
L461
P418
R360
K385
Q122
L198
T228
P426
G399
E196
F223
R200
F296
P316
V222I292 Y226
I302V240
H169
Y189 Q122
H160
N371
V374 I377
E378H358
P359
R360
E381
L382N386
K385
P418
F451
F354
L453
R332
Y389
F487
Fig. S4. The Air2p-interaction surface of Trf4p is conserved in eukaryotes. As in Fig. 3, but larger.
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A
B
Fig. S5. Availability of surfaces in the fourth and fifth zinc knuckle modules for RNA binding. (A) Residues corresponding to those in nucleocapsid zincknuckles that are involved in binding single-stranded RNAs are colored orange. Residues in the fourth zinc knuckle are accessible at the surface of theTrf4p∕Air2pZK4-5 complex and could interact with RNAs in the same way as corresponding residues in the nucleocapsid proteins. The view is in stereo.(B) As in A, but a different orientation.
lane 1 3 5 7
A10 C10 G10 U10
BA
lane MW 1 3 5 7 9 11
0 +1 +3 +5 +7 +10
3’ overhang length
110124
147190242320
489,501692
900, 1114
67
37
19
2634
Fig. S6. RNA substrates of the full-length Trf4p/Air2p complex. Experiments are as in Fig. 4, but preparations of full-length complex were used. Due tosignificant sample degradation (Fig. S1), the amount of full-length protein used was difficult to quantitate. (A) Polyadenylation of 5′ end-labeled oligomersA10, C10, G10, or U10 (30 fmol) by Trf4p/Air2p. No protein was added in lanes 1, 3,5, or 7. (B) Polyadenylation assay, where Trf4p/Air2p was mixed with RNAduplexes (30 fmol) containing no overhang (lanes 1 and 2), or 3′- A-overhangs of length indicated (lanes 3–12). Duplex sequences were based on the acceptorstem of tRNAAla, as in Fig. 4C. Longer extensions were added only when the 3′ overhang was three nucleotides or longer. No protein was added in lanes 1, 3, 5,7, 9, or 11.
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F404 F404
W409W409 W409W409
F404F404
H8 H8
H9 H9
H5H5
A B
C DZK1 ZK1
E F
Fig. S7. Electron density maps, in stereo. (A) This region of the map, corresponding to a part of the Trf4p central domain, was poorly defined in the initialsingle anomalous wavelength dispersion maps. The experimental phases were combined with phases calculated from a partial model, in which this portion ofTrf4p was not included. The resultingmap is shown here, contoured at 1.0 sigma, andwas used for model building. (B) The same region in a simulated-annealed(T ¼ 3;000 K) composite omit map contoured at 1.0 sigma. The mapwas calculated in the CNS software suite. (C) The composite omit map showing a portion ofthe central domain. (D) The same view, but with the final model included. (E) Ribbons diagram of the Trf4p∕Air2pZK4-5 complex indicating the variation in Bfactors across the molecule (red, high; blue, low).
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Table S1. Summary of crystallographic data and refinement
Data collection
Anomalous data (3 crystals) Data for refinement (single crystal)Spacegroup P321 P321Cell constants, Å a ¼ b ¼ 92.25, c ¼ 103.60 A ¼ b ¼ 92.434, c ¼ 103.20Wavelength, Å 1.28215 1.28215Resolution (last shell), Å 25.0-3.0 (3.11-3.00) 25.0-2.7 (2.80-2.70)No. unique reflections (last shell) 19704 (1980) 14370 (1413)
[Bijvoet reflections separate] [Bijvoet reflections merged]Completeness (last shell), % 100 (100) 100 (100)Redundancy (last shell) 15.0 (15.3) 10.8 (10.9)Rsym (last shell), % 10.9 (33.2) 9.2 (64.6)hIi∕hσi (last shell) 41.9(12.8) 39.6(4.3)
Refinement
Resolution (last shell), Å 20-2.70Rwork∕Rfree, % 19.35∕25.81Total no. residues 386Total no. waters 58
rmsd deviationsbond lengths, Å 0.006bond angles, ° 1.004dihedral angles, ° 19.151
Average B factors, Å2
All proteins 65.98Main chain/side chain 62.70∕69.15
Trf4p 59.96Main chain/side chain 56.20∕63.53
Air2p 90.68Main chain/side chain 88.50∕92.88
Ramachandran plotResidues in most favored region, % 82.8Additionally allowed, % 17.2Generously allowed or not allowed, % None
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