The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity

Abstract

The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

ARTICLE

Received 13 Mar 2012 | Accepted 7 Jun 2012 | Published 10 Jul 2012 DOI: 10.1038/ncomms1947

The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc nger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity

Kehui Xiang1, James L. Manley1 & Liang Tong1

1 Department of Biological Sciences, Columbia University, New York, 10027, USA. Correspondence and requests for materials should be addressed to L.T. (email: [email protected]).

NATURE COMMUNICATIONS | 3:946 | DOI: 10.1038/ncomms1947 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1947

The activity of RNA polymeraes II (Pol II) is regulated by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit15, which contains the consensus heptapep-

tide repeat Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (YSPTSPS). While all the hydroxyl groups in this repeat can become phosphorylated, phosphorylation of Ser2 and Ser5 has received the most attention to date. For example, phosphorylated Ser5 (pSer5) is found at the promoter and during the initiation and early stages of Pol II transcription, and it mediates the recruitment of the 5-end capping machinery and other factors. pSer5 is then dephosphorylated, and Ser2 becomes phosphorylated during the elongation and termination stages of transcription, which also facilitates 3-end processing of the pre-mRNA transcript.

In keeping with the importance of Ser2 and Ser5 phosphorylation, the kinases and phosphatases involved have been well studied5. The phosphatase Fcp1 is present along the length of transcribed genes, and can dephosphorylate both pSer2 and pSer5, but prefers pSer26,7. Ssu72 is a known CTD pSer5 phosphatase8,9, although it is localized primarily near the 3-end of genes. Ssu72 prefers the cis conguration of the pSer5-Pro6 peptide bond10,11, and it helps to couple transcription and polyadenylation10. Scp1 is another pSer5 phosphatase in metazoans, and it may function near the 5 end of certain genes1214.

It was recently reported that the yeast protein Rtr1 (regulator of transcription15) has Pol II CTD pSer5 phosphatase activity and is required for pSer5 dephosphorylation in the early stage of transcription16. The human homologue of Rtr1, RPAP2 (RNA Pol II-associated protein 217), has also been reported to have CTD pSer5 phosphatase activity18. These proteins are poorly conserved overall (Fig. 1), but they share a motif with three strictly conserved Cys residues and another residue conserved as His (in most fungi) or Cys (in animals and S. pombe, Fig. 1), suggestive of a zinc nger motif15. However, the spacing among these residues (C-X4-C-Xn-C-X3-H/C, where n ranges from 30 to 50) as well as the sequences of Rtr1 homologues are distinct from known zinc nger proteins.

In this study, we report the crystal structure of Rtr1. It reveals a new type of zinc nger protein, in fact distinct from other known protein structures in general. However, the structure lacks an apparent active site, and the zinc ion probably has a structural role, likely stabilizing the overall structure of the protein. Moreover, extensive eorts to demonstrate CTD phosphatase activity for Rtr1 from several dierent fungal species as well as for human RPAP2 were unsuccessful, in contrast to the earlier reports but consistent with the structural observations. Therefore, Rtr1/RPAP2 itself is unlikely

a phosphatase, but perhaps has a non-catalytic role in CTD dephosphorylation. The identity of the pSer5 phosphatase in the early stage of Pol II transcription remains to be determined.

ResultsOverall structure of Rtr1. We have determined the crystal structure of Kluyveromyces lactis Rtr1 (KlRtr1) at 2.5 resolution (Table 1, Supplementary Fig. S1). The bacterial growth media was supplemented with zinc during protein expression, and the puried protein had nearly stoichiometric amounts of zinc19. Fluorescence scans were carried out on the crystal at the absorption edges of Zn, Ni, Co and Fe, and anomalous signals were observed only at the Zn edge, which were used to solve the structure. The full-length protein (residues 1211) was used for crystallization, but only residues 1152 were observed in the structure. SDS gels of the crystals showed that the C-terminal segment of KlRtr1 was removed by proteolysis during crystallization (Supplementary Fig. S2). The sequence conservation for these C-terminal residues is much weaker among Rtr1 and RPAP2 homologues (Fig. 1).

The structure of KlRtr1 (residues 1152) contains ve anti- parallel -helices (AE, Fig. 2a). A long loop (residues 70112) connects helices D and E, and the three strictly conserved Cys residues (73, 78 and 111) are located in this loop. Residues 88100 at the tip of this loop are disordered in the current structure (Fig. 2a), and this loop may have also been proteolysed in some of the Rtr1 molecules in the crystal (Supplementary Fig. S2). The fourth ligand to the zinc ion, His115 or Cys, is in the rst turn of helix E. This helix is followed by another long loop (residues 126152) that wraps around one side of the structure (Fig. 2a).

We also obtained crystals of Ashbya gossypii Rtr1 (AgRtr1), although the best diraction data set extended only to 3.5 resolution. By combining the structural information from KlRtr1 and primary phase information from Zn anomalous signals, we were able to observe another helix in the C-terminal region of AgRtr1, likely equivalent to residues 154166 of KlRtr1 (Fig. 1). This helix is projected away from the rest of the protein and is stabilized by crystal packing interactions in the AgRtr1 crystal (Supplementary Fig. S3), suggesting that the C-terminal region of Rtr1 may function independently of the N-terminal region.

Rtr1 is a new type of zinc nger protein. The zinc ion is coordinated in a tetrahedral fashion by the four conserved ligands (Fig. 2a and b). The zinc-binding site is located near the surface, but there are no prominent features in this region of the protein (see below). The main-chain carbonyl oxygen atoms of the rst two ligands,

S. S. A B C DKlRtr1 1 MATIEEIKEVVLKPYTNHRQLTIREVETISINLIDLLITKDVKDARTMKYISRFLTKQDYADLVQERNLVKRCGYPLCSKSQ

ScRtr1 1 MATIEDIKETALIPFQKHRQLSMHEAEVITLEIIGLLCDSECKDEKTLKYLGRFLTPDMYQDLVDERNLNKRCGYPLCGKSPAgRtr1 1 MATIEDIQKQVLVAYQQHRQLSLKEADLLTVELLELLSQTECTP-ETLKYVGRFFTKTTYSDLIDERNLNKLCGYPLCGQSQHsRPAP2 29 TSTLKQEDASKRKAELEAAVRKKIEFERKALHIVEQLLEENITEEFLMECG-RFITPAHYSDVVDERSIVKLCGYPLCQKKL

S. S. EKlRtr1 83 ARVR-Dpfadvqmtn-flrQNNPYAYLTEYCTKAHFRCSQFYQFQLSDEALFARVGVHL------DDYEPPSEIQLLEevl

ScRtr1 83 ERIR-DPFSMNDTTKKFLLENNPYAYLSHYCSKFHFRCSQFYQVQLSDEALFARTGVHLFEDPEQDKHDIDFKVTLFEELLAgRtr1 82 GRIR-DPYENRRVSN-FLTQNNPYKYLTSFCSKLHFRCSQFYQVQLSDEALFARVGVHL-------DDYEAGRVLLLEEAMHsRPAP2 110 GIVPKQKYKISTKTN----KVYDITERKSFCSNFCYQASKFFEAQIPKTPVWVREEERH-----------PDFQLLKEEQS

S. S. F

S. S. F

. . . . . . . .

. . . . . . .

. . . . . .KlRtr1 156 ---aresdvkqmirgmtdlkladdd-sre----emerdisdmladikiveinepniigdlgrdr 211

ScRtr1 163 REKASEEDIKSLISGLKKLGLNPDSGTTEKDDTELEDDLSKWLAQIKIVENDNPSILGDFTRED 226AgRtr1 154 ---AEERDLRTMIRGMNGLSLGENG-AGD----ELQKDLSEWLSEVRIVENERPPLLGDLMKE 208HsRPAP2 176 GHSGEEVQLCSKAIKTSDIDNPSHFEKQYESSSSSTHSDSSSDNEQDFVSSILPGNRPNSTNIRP 240 (612)

Figure 1 | Sequence conservation among Rtr1 and RPAP2 homologues. Sequence alignment of full-length K. lactis, S. cerevisiae and A. gossypii Rtr1 (KlRtr1, ScRtr1, AgRtr1) and human RPAP2 (HsRPAP2, residues 1240 only, out of 612 residues total) is shown. The four zinc ligand residues are shown in orange. Residues conserved among 11 fungal Rtr1 homologues (excluding the second Rtr1 homolog in S. cerevisiae) or 8 animal RPAP2 homologues are shown in magenta. Residues conserved among fungal Rtr1 and animal RPAP2 homologues are shown in blue. Residues not observed in the crystal structure of KlRtr1 are shown in lower-case italic. The secondary structure elements (S.S.) are labelled.

NATURE COMMUNICATIONS | 3:946 | DOI: 10.1038/ncomms1947 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1947

ARTICLE

Table 1 | Summary of crystallographic information.

K. lactis Rtr1 A. gossypii Rtr1

Data collection

Space group P212121 I422Cell dimensionsa, b, c () 45.2, 88.7, 94.9 115.4, 115.4, 125.4 , , () 90, 90, 90 90, 90, 90

Peak Inection Remote Peak Wavelength () 1.2833 1.2836 1.2652 1.2830 Resolution range ()* 502.5 (2.62.5) 502.5 (2.62.5) 502.5 (2.62.5) 503.5 (3.63.5) Rmerge (%) 5.5 (35.5) 5.6 (42.5) 5.6 (37.9) 6.5 (43.3)

I/I 21.9 (4.0) 21.3 (3.5) 21.1 (3.5) 18.4 (2.4) Completeness (%) 99 (100) 99 (100) 99 (99) 99 (100) Redundancy 3.7 (3.8) 3.7 (3.8) 3.7 (3.6) 4.4 (4.7)

Renement

Resolution () 502.5 No. of reections 12,890 Rwork/Rfree (%) 22.2/27.2

No. of atoms

Protein 2,243 Ligand/ion 2 Water 32

B-factors

Protein 57.9 Ligand/ion 75.6 Water 55.5

R.m.s. deviations

Bond lengths () 0.017 Bond angles () 1.6

Abbreviations: MAD, multiple-wavelength anomalous diffraction; R.m.s. deviation, root-mean-square deviation; SAD, single-wavelength anomalous diffraction.

*The numbers in parentheses are for the highest resolution shell. One crystal was used for the K. lactis MAD data set, and one crystal for the A. gossypii SAD data set.

Cys73 and Cys78, are hydrogen bonded to the guanidinium group of Arg67, one of the few other conserved residues among these proteins (Fig. 1). The hydrogen bonds stabilize the two Cys residues as well as the loop connecting them, which is hydrophobic in nature and contributes to the formation of the hydrophobic core of Rtr1 (Fig. 2a). The zinc ion therefore seems to have a structural role, likely stabilizing the overall conformation of Rtr1.

We have identied Rtr1 as a new type of zinc nger protein. A search through the Protein Data Bank with DaliLite20 did not identify any close structural homologues, with the highest Z score being 3.3. The overall fold of Rtr1 is somewhat reminiscent of the HEAT repeats, although the positions of the helices are dierent compared with these repeats. The topology of the zinc ligands in Rtr1, with the rst three located in loops and the last one in the beginning of a helix, has some similarity to the A20 family of zinc nger proteins21, as illustrated by the structure of Rabex-5, a guanine nucleotide exchange factor for Rab5 that also binds monoubiquitin22,23. The overall conformations of the protein backbone near the rst two zinc ligands are similar between Rtr1 and Rabex-5 (Fig. 2c). On the other hand, the loop connecting them, and especially the loop connecting to the third ligand, has dierent numbers of residues (the zinc ligands in Rabex-5 have the motif C-X3-C-X11-C-X2-C). In addition, the orientation of the helix containing the fourth ligand diers by nearly 90 between the two structures. Finally, the zinc-binding site in Rtr1 is part of a much larger structure, with 150 residues (Fig. 2a), whereas the zinc nger domain of Rabex-5 contains only 35 residues.

Puried Rtr1/RPAP2 lack detectable CTD phosphatase activity. We next attempted to demonstrate Pol II CTD pSer5 phosphatase

activity with our puried protein samples of His-tagged Rtr1 from K. lactis, A. gossypii, S. cerevisiae and several other fungal species. To test the possibility that Rtr1 in the absence of zinc or in complex with a dierent metal ion could be catalytically active, we used protein samples puried from E. coli cells that were grown without additional zinc in the medium. The zinc occupancy ranged between 10 and 80%, but other metal ions could be present in these samples. We also expressed and puried residues 1332 of human RPAP2, as His-tagged and GST-fusion proteins, and full-length RPAP2 as a GST-fusion protein18. Despite extensive eorts and using a large collection of dierent substrates (Supplementary Table S1), we failed to detect CTD pSer5 phosphatase activity for Rtr1 and RPAP2 under any of the conditions tested, while robust activity was observed with Ssu72. The substrates used include a CTD peptide Ser7-Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7-Tyr1-Ser2 phosphorylated at the Ser5, Ser5 and Ser7, or Ser2, Ser5 and Ser7 positions, as it has been shown that RPAP2 requires Ser7 phosphorylation to interact with the CTD18. These assays monitored the release of free phosphate from the reactions. We also used as the substrate the entire CTD puried as a GST-fusion protein10 and either phosphorylated with HeLa cell nuclear extract or puried Cdk7. The reactions were monitored by western blotting with an antibody specic for pSer5. We again failed to detect activity with multiple Rtr1/RPAP2 samples and under a variety of conditions (Fig. 3a and b and results not shown). In addition, we soaked Rtr1 crystals with CTD phosphopeptides, but were not able to identify any binding based on the crystallographic analysis.

The structure of Rtr1 lacks an apparent active site. The lack of phosphatase activity for Rtr1 is consistent with our structure, which

NATURE COMMUNICATIONS | 3:946 | DOI: 10.1038/ncomms1947 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1947

pH=5.5

0.2 1 0.2 1 5 20 1 1 (g)

a

87

101

a

pH=6.5

101

Ssu72

0

KIRtr1 KIRtr1

Ssu72

87

Zn

C111

Zn

100 kD

pSer5

(H14)

C73

GSTCTD (HeLa NE)

D

H115

60

D

152 E

C78

E

152

1

C

b

1

RPAP2 1332 NHis (5 g)

RPAP2 1332 GST (5 g)

RPAP2 (5 g)

B

B

Ssu72 (0.5 g)

AgRtr1 (5 g)

ScRtr1 (5 g)

C

A

A

pSer5

(H14)

b c

100 kD

Control

GSTCTD (Cdk7)

Figure 3 | Rtr1 and RPAP2 failed to show phosphatase activity with GST CTD as the substrate. (a) With the GSTCTD substrate phosphorylated by HeLa cell nuclear extract, KlRtr1 shows no signs of activity under various conditions. (b) Phosphatase activity of Rtr1 from different species and human RPAP2 was tested against GSTCTD substrate phosphorylated by the Cdk7 complex. Ssu72 was used as a positive control in both cases.

C111

H115

C73

C111/C35

C73/C19

C78/C23

Zn

H115/C38

C78

Zn

Figure 2 | Rtr1 is a new type of zinc nger protein. (a) Schematic drawing of the structure of K. lactis Rtr1. The zinc atom is shown as a sphere (in orange), and its four ligands are shown as stick models (in cyan). The two views are related by a rotation of ~60 around the vertical axis. (b) Simulated annealing omit FoFc electron density for the zinc atom

and its ligands in the structure of KlRtr1 at 2.5 resolution, contoured at 2.5. (c) Overlay of the zinc-binding site of K. lactis Rtr1 (in colour) and the zinc nger domain of Rabex-5 (in grey). The superposition is based on the rst two zinc ligands. All the structure gures were produced with PyMOL (www.pymol.org).

does not indicate the presence of an active site. An analysis of the surface features that are conserved among Rtr1 and RPAP2 homologues does not reveal any pockets that are lined with conserved residues and could function as an active site (Fig. 4). The C-terminal segment that is missing in the current structure is unlikely to form an active site, owing to its poor conservation (Fig. 1). The zinc-binding site cannot be an active site either, as the zinc ion is tetrahedrally coordinated by the four ligands and is not capable of binding and activating a water molecule for hydrolytic activity. Moreover, the zinc-binding site corresponds to a small protrusion on the surface of Rtr1 (Fig. 4), and there is no pocket nearby that could bind the CTD substrate. Mutation of one of the Zn ligands in ScRtr1 (Cys73) was reported to abolish the dephosphorylation of pSer5 in yeast cells16. It is more likely that this mutation disrupted zinc binding and thereby the overall structure of the protein. We do not know why phosphatase activity was observed in vitro in the earlier reports16,18. A co-purifying protein with this activity might be a possibility.

Discussion

If Rtr1/RPAP2 is not a phosphatase, what then is its function in Pol II transcription? Rtr1 is found primarily in the cytoplasm, but can shuttle into the nucleus15. Recent reports suggest that Rtr1 and RPAP2 may have an important role in the assembly and transport of Pol II from the cytoplasm into the nucleus2426. These and other1518

data establish that Rtr1 interacts with Pol II, and therefore it is likely that Rtr1 could mediate interactions among various proteins in the Pol II complex. Given the evidence that Rtr1/RPAP2 is important for pSer5 dephosphorylation in vivo16,18, an attractive possibility is that Rtr1/RPAP2 is required for recruitment and/or activation of the actual phosphatase that dephosphorylates pSer5 during the early stages of Pol II transcription. The identity of that phosphatase remains to be determined.

Methods

Protein expression and purication. Full-length Rtr1 from K. lactis, A. gossypii, S. cerevisiae and the N-terminal region (residues 1332) of human RPAP2 were cloned into the pET28a vector (Novagen) and overexpressed in E. coli BL21 (DE3) Star cells at 20 C by the addition of 0.5 mM isopropyl--d-thiogalactopyranoside at OD600 of 0.6. To help the zinc enrichment in the protein, 0.1 mM ZnSO4 was added to the culture 1 h before induction. The expression constructs introduced hexa-histidine tags at the N terminus of the proteins.

The soluble proteins were puried by Ni-NTA (Qiagen) with the elution buer containing 20 mM Tris (pH 7.5), 200 mM NaCl and 250 mM imidazole, followed by gel ltration (Sephacryl S-300, GE Healthcare) chromatography in a running buer of 20 mM Tris (pH 7.5), 200 mM NaCl and 2 mM DTT. The proteins were concentrated to 20 mg ml 1 in a buer containing 20 mM Tris (pH 7.5), 200 mM NaCl, 2 mM DTT and 5% (v/v) glycerol, ash frozen in liquid nitrogen and stored at 80 C.

The GST fusion RPAP2 proteins were made by cloning the full-length human RPAP2 or the N-terminal region (residues 1332) into pGEX-4T-3 (GE Life Sciences) vector and overexpressed in E. coli BL21 (DE3) Star cells with the same protocol as mentioned above. The proteins were puried by glutathione Sepharose 4 Fast Flow (GE Healthcare) with 20 mM reduced glutathione in a buer of 20 mM Tris (pH 7.5), 200 mM NaCl and 5% (v/v) glycerol.

Protein crystallization. Crystals of KlRtr1 were obtained with the sitting-drop vapour diusion method at 20 C. The reservoir solution contained 100 mM CHES (pH 9.5) and 20% (w/v) PEG8000. The crystals belong to space group P212121, and there are two molecules in the asymmetric unit. Crystals of AgRtr1 were obtained with the sitting-drop vapour diusion method at 20 C. The reservoir contained 120 mM Tris (pH 7.0) and 16% (v/v) ethanol. The crystals belong to space group I422, and there is one molecule in the asymmetric unit. The crystals were cryo- protected by the respective reservoir solutions supplemented with 25% (v/v) ethylene glycol and ash frozen in liquid nitrogen for data collection at 100 K.

Data collection and structure determination. X-ray diraction data were collected on an ADSC charge-coupled device at the X29A beamline of National

NATURE COMMUNICATIONS | 3:946 | DOI: 10.1038/ncomms1947 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1947

ARTICLE

Zn

180

E151 Q125 E129

E66 L56

R134

E25

Figure 4 | The structure of Rtr1 does not show evidence for an active site. Molecular surface of K. lactis Rtr1 (front and back views), coloured based on sequence conservation36, from highly conserved (purple) to poorly conserved (blue) residues. The highly conserved surface patches are labelled.

Synchrotron Light Source (NSLS). The diraction images were processed and scaled with the HKL package27. The atomic coordinates of the KlRtr1 structure have been deposited in the Protein Data Bank under the accession code 4FC8.

Multiple-wavelength anomalous diraction data sets were collected on the KlRtr1 crystal to 2.5 resolution, at the zinc absorption edge (inection point, 1.2836 ), peak (1.2833 ) and remote (1.2652 ) wavelengths. The data processing statistics are summarized in Table 1. The Zn sites were located with the program BnP28. Reection phases were calculated using the program SOLVE/RESOLVE29. The complete atomic model was t into the electron density with the programs O30 and Coot31. The structure renement was carried out with the programs CNS32 and Refmac33, against the data set at the peak wavelength. The statistics on the structure renement are summarized in Table 1. For the nal atomic model, 95.2% of the residues are in the most favoured region of the Ramachandran plot, 4.8% in additional allowed regions and none in the disallowed region.

For the AgRtr1 crystal, a single-wavelength anomalous diraction data set to

3.5 resolution was collected at the zinc peak wavelength. An electron density map was obtained based on the single-wavelength anomalous diraction data, which showed clear indications for several helices. The atomic model of KlRtr1 couldbe readily positioned into the density, revealing an extra helix in the C-terminal region in the AgRtr1 structure. Renement of this structure model was not carried out due to the limited resolution.

CTD peptide phosphatase assays. Reaction mixtures (25 l) in the standard phosphatase condition (50 mM Bis-Tris (pH 6.5), 20 mM KCl, 10 mM MgCl2

and 5 mM DTT) containing 500 M CTD peptide, Rtr1, RPAP2 or Ssu72 at 1 M, 5 M or 20 M concentration were incubated at 30 C. Time-point samples were taken and quenched by adding 0.5 ml of malachite green reagent (BIOMOL Research Laboratories, Plymouth Meeting, PA). Phosphate release was determined by measuring A620 and comparing it with a phosphate

standard curve.

CTD phosphatase assay. Puried GSTCTD fusion protein was phosphorylated in vitro by HeLa cell nuclear extract as described34, or by Cdk7 complex as described35. CTD phosphatase assays were performed in a total volume of 20 l in the standard phosphatase condition containing 200 ng phosphorylated GSTCTD and indicated amount of Rtr1, RPAP2 or Ssu72. Reactions were incubated for 1 h at 30 C, stopped by adding 5 l 5 SDS loading buer and 2.5 l from each reaction was resolved on an 8% SDSPAGE gel. pSer5 level was detected by western blot using H14 antibody (Covance).

All CTD peptide phosphatase assays and CTD phosphatase assays were additionally performed in dierent conditions by varying pH (5.58.5) or KCl concentration (20500 mM) or additives (110 mM ZnCl2 or MnCl2 or MgCl2)

based on the standard phosphatase condition.

References

1. Komarnitsky, P., Cho, E. J. & Buratowski, S. Dierent phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Develop. 14, 24522460 (2000).

2. Schroeder, S. C., Schwer, B., Shuman, S. & Bentley, D. Dynamic association of capping enzymes with transcribing RNA polymerase II. Genes Develop. 14, 24352440 (2000).

3. Meinhart, A., Kamenski, T., Hoeppner, S., Baumli, S. & Cramer, P.

A structural perspective of CTD function. Genes Develop. 19, 14011415 (2005).

4. Phatnani, H. P. & Greenleaf, A. L. Phosphorylation and functions of the RNA polymerase II CTD. Genes Develop. 20, 29222936 (2006).

5. Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol. Cell 36, 541546 (2009).

6. Hausmann, S., Erdjument-Bromage, H. & Shuman, S. Schizosaccharomyces pombe carboxylterminal domain (CTD) phosphatase Fcp1: distributive mechanism, minimal CTD substrate, and active site mapping. J. Biol. Chem. 279, 1089210900 (2004).

7. Ghosh, A., Shuman, S. & Lima, C. D. The structure of Fcp1, an essential RNA polymerase II CTD phosphatase. Mol. Cell 32, 478490 (2008).

8. Krishnamurthy, S., He, X., Reyes-Reyes, M., Moore, C. L. & Hampsey, M. Ssu72 is an RNA polymerase II CTD phosphatase. Mol. Cell 14, 387394 (2004).

9. Hausmann, S., Koiwa, H., Krishnamurthy, S., Hampsey, M. & Shuman, S. Dierent strategies for carboxyl-terminal domain (CTD) recognition by serine 5-specic CTD phosphatases. J. Biol. Chem. 280, 3768137688 (2005).

10. Xiang, K. et al. Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex. Nature 467, 729733 (2010).

11. Werner-Allen, J. W et al. cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72. J. Biol. Chem. 286, 57175726 (2011).

12. Yeo, M., Lin, P. S., Dahmus, M. E. & Gill, G. N. A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5. J. Biol. Chem. 278, 2607826085 (2003).13. Kamenski, T., Heilmeier, S., Meinhart, A. & Cramer, P. Structure and mechanism of RNA polymerase II CTD phosphatases. Mol. Cell 15, 399407 (2004).

14. Zhang, Y. et al. Determinants for dephosphorylation of the RNA polymerase II C-terminal domain by Scp1. Mol. Cell 24, 759770 (2006).

15. Gibney, P. A., Fries, T., Bailer, S. M. & Morano, K. A. Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins. Eukaryotic Cell 7, 938948 (2008).

16. Mosley, A. L. et al. Rtr1 is a CTD phosphatase that regulates RNA polymerase II during the transition from serine 5 to serine 2 phosphorylation. Mol. Cell 34, 168178 (2009).

17. Jeronimo, C. et al. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol. Cell 27, 262274 (2007).

18. Eglo, S., Zaborowska, J., Laitem, C., Kiss, T. & Murphy, S. Ser7 phosphorylation of the CTD recruits the RPAP2 Ser5 phosphatase to snRNA genes. Mol. Cell 45, 111122 (2012).

19. McCall, K. A. & Fierke, C. A. Colorimetric and uorimetric assays to quantitate micromolar concentrations of transition metals. Anal. Biochem. 284, 307315 (2000).

20. Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 27802781 (2008).

21. Gamsjaeger, R., Liew, C. K., Loughlin, F. E., Crossley, M. & Mackay, J. P. Sticky ngers: zinc-ngers as protein-recognition motifs. Trends Biochem. Sci. 32, 6370 (2007).

22. Penengo, L. et al. Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin. Cell 124, 11831195 (2006).

23. Lee, S. et al. Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5. Nature Struct. Mol. Biol. 13, 264271 (2006).

24. Boulon, S. et al. HSP90 and its R2TP/prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol. Cell 39, 912924 (2010).

25. Czeko, E., Seizl, M., Augsberger, C., Mielke, T. & Cramer, P. Iwr1 directs RNA polymerase II nuclear import. Mol. Cell 42, 261266 (2011).

26. Corden, J. Going nuclear: transcribers in transit. Mol. Cell 42, 143145 (2011).

27. Otwinowski, Z. & Minor, W. Processing of X-ray diraction data collected in oscillation mode. Methods Enzymol. 276, 307326 (1997).

28. Weeks, C. M et al. Automatic solution of heavy-atom substructures. Methods Enzymol. 374, 3783 (2003).

29. Terwilliger, T. C SOLVE and RESOLVE: Automated structure solution and density modication. Methods Enzymol. 374, 2237 (2003).

30. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Cryst. A47, 110119 (1991).

31. Emsley, P. & Cowtan, K. D. Coot: model-building tools for molecular graphics. Acta Cryst. D60, 21262132 (2004).

32. Brunger, A. T. et al. Crystallography & NMR System: A new soware suite for macromolecular structure determination. Acta Cryst. D54, 905921 (1998).

NATURE COMMUNICATIONS | 3:946 | DOI: 10.1038/ncomms1947 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1947

33. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Renement of macromolecular structures by the maximum-likelihood method. Acta Cryst. D53, 240255 (1997).

34. Hirose, Y. & Manley, J. L. RNA polymerase II is an essential mRNA polyadenylation factor. Nature 395, 9396 (1998).

35. Glover-Cutter, K. et al. TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II. Mol. Cell. Biol. 29, 54555464 (2009).

36. Armon, A., Graur, D. & Ben-Tal, N. ConSurf: an algorithmic tool for the identication of functional regions in proteins by surface mapping of phylogenetic information. J. Mol. Biol. 307, 447463 (2001).

Acknowledgements

We thank Amber Mosley and Lawrence Chasin for discussions; Stephane Larochelle and Robert P. Fisher for Cdk7 baculovirus; Shona Murphy for sharing a preprint on RPAP2; Neil Whalen, Stuart Myers and Howard Robinson for setting up the X29A beamline at the NSLS. This research is supported by grants from the NIH to LT (GM077175) and JLM (GM028983).

Author contributions

K.X. carried out all experiments. J.L.M. analysed the phosphatase data. L.T. conceived the project, analysed the data and wrote the paper. All authors commented on and contributed to the manuscript.

Additional information

Accession codes: The atomic coordinates of the KlRtr1 structure have been deposited in the Protein Data Bank under the accession code 4FC8.

Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications

Competing nancial interests: The authors declare no competing nancial interests.

Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/

How to cite this article: Xiang, K. et al. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity. Nat. Commun. 3:946 doi: 10.1038/ncomms1947 (2012).

NATURE COMMUNICATIONS | 3:946 | DOI: 10.1038/ncomms1947 | www.nature.com/naturecommunications

2012 Macmillan Publishers Limited. All rights reserved.