Structure and Carboxyl-terminal Domain (CTD) Binding of the Set2 SRI Domain That Couples Histone H3 Lys36 Methylation to Transcription
2005; Elsevier BV; Volume: 281; Issue: 1 Linguagem: Inglês
10.1074/jbc.c500423200
ISSN1083-351X
AutoresErika Vojnic, Bernd Simon, Brian D. Strahl, Michael Sattler, Patrick Cramer,
Tópico(s)RNA modifications and cancer
ResumoDuring mRNA elongation, the SRI domain of the histone H3 methyltransferase Set2 binds to the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. The solution structure of the yeast Set2 SRI domain reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. NMR titration shows that the SRI domain binds an Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues, whereas a single CTD repeat is insufficient for binding. Residues that show strong chemical shift perturbations upon CTD binding cluster in two regions. Both CTD tyrosine side chains contact the SRI domain. One of the tyrosines binds in the region with the strongest chemical shift perturbations, formed by the two NH2-terminal helices. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in bacterial σ factors (domain σ2 in σ70). During mRNA elongation, the SRI domain of the histone H3 methyltransferase Set2 binds to the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. The solution structure of the yeast Set2 SRI domain reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. NMR titration shows that the SRI domain binds an Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues, whereas a single CTD repeat is insufficient for binding. Residues that show strong chemical shift perturbations upon CTD binding cluster in two regions. Both CTD tyrosine side chains contact the SRI domain. One of the tyrosines binds in the region with the strongest chemical shift perturbations, formed by the two NH2-terminal helices. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in bacterial σ factors (domain σ2 in σ70). Gene transcription by RNA polymerase II (Pol II) is physically and functionally coupled to other nuclear events, most notably mRNA processing (1Dahmus M.E. Biochim. Biophys. Acta. 1995; 1261: 171-182Crossref PubMed Scopus (109) Google Scholar, 2Palancade B. Bensaude O. Eur. J. Biochem. 2003; 270: 3859-3870Crossref PubMed Scopus (202) Google Scholar, 3Sims III, R.J. Mandal S.S. Reinberg D. Curr. Opin. Cell Biol. 2004; 16: 263-271Crossref PubMed Scopus (150) Google Scholar, 4Zorio D.A. Bentley D.L. Exp. Cell Res. 2004; 296: 91-97Crossref PubMed Scopus (116) Google Scholar, 5Proudfoot N. Curr. Opin. Cell Biol. 2004; 16: 272-278Crossref PubMed Scopus (239) Google Scholar, 6Maniatis T. Reed R. Nature. 2002; 416: 499-506Crossref PubMed Scopus (928) Google Scholar, 7Meinhart A. Kamenski T. Hoeppner S. Baumli S. Cramer P. Genes Dev. 2005; 19: 1401-1415Crossref PubMed Scopus (261) Google Scholar). 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Dev. 2001; 15: 3319-3329Crossref PubMed Scopus (340) Google Scholar). carboxyl-terminal domain RNA polymerase II Set2 Rpb1-interacting nuclear Overhauser effect nuclear Overhauser and exchange spectroscopy Protein Data Bank. Recently it emerged that transcription is also coupled to the alteration of chromatin structure. The histone methyltransferases Set1 and Set2, which catalyze methylation of histone H3 lysines Lys4 and Lys36, respectively, are associated with Pol II during elongation (reviewed in Refs. 14Gerber M. Shilatifard A. J. Biol. Chem. 2003; 278: 26303-26306Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar and 15Hampsey M. Reinberg D. Cell. 2003; 113: 429-432Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Histone methylation apparently controls newly initiated Pol II, and two phases of histone H3 methylation can be distinguished after transcription initiation (16Morillon A. Karabetsou N. Nair A. Mellor J. Mol. Cell. 2005; 18: 723-734Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Set1 association with Pol II is mediated by the Paf complex, which occurs in promoter regions, and depends on Ser5 phosphorylation of the CTD (17Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar, 18Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar). In contrast, Set2 directly interacts with the phosphorylated CTD of Pol II and is observed throughout the coding region of genes (17Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar, 18Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar, 19Strahl B.D. Grant P.A. Briggs S.D. Sun Z.W. Bone J.R. Caldwell J.A. Mollah S. Cook R.G. Shabanowitz J. Hunt D.F. Allis C.D. Mol. Cell. Biol. 2002; 22: 1298-1306Crossref PubMed Scopus (433) Google Scholar, 20Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar). Set2 recruitment to Pol II requires the CTD kinase CTDK-I that phosphorylates Ser2 residues in the CTD (17Krogan N.J. Kim M. Tong A. Golshani A. Cagney G. Canadien V. Richards D.P. Beattie B.K. Emili A. Boone C. Shilatifard A. Buratowski S. Greenblatt J. Mol. Cell Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (519) Google Scholar, 18Ng H.H. Robert F. Young R.A. Struhl K. Mol. Cell. 2003; 11: 709-719Abstract Full Text Full Text PDF PubMed Scopus (857) Google Scholar, 20Xiao T. Hall H. Kizer K.O. Shibata Y. Hall M.C. Borchers C.H. Strahl B.D. Genes Dev. 2003; 17: 654-663Crossref PubMed Scopus (326) Google Scholar, 21Li B. Howe L. Anderson S. Yates III, J.R. Workman J.L. J. Biol. Chem. 2003; 278: 8897-8903Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Set2 interacts with the Pol II CTD via a novel domain, the Set2 Rpb1-interacting (SRI) domain (22Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar, 23Phatnani H.P. Jones J.C. Greenleaf A.L. Biochemistry. 2004; 43: 15702-15719Crossref PubMed Scopus (97) Google Scholar). The SRI domain of S. cerevisiae comprises the COOH-terminal residues 619–718 of Set2 (22Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar). In vitro, the yeast Set2 SRI domain binds specifically and with high affinity to the CTD doubly phosphorylated at Ser2 and Ser5 (22Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar). In vivo, deletion of the Set2 SRI domain abolishes H3 Lys36 methylation and impairs transcription elongation (22Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar), suggesting that the SRI domain is responsible for coupling transcription to histone methylation by Set2. Here we report the solution structure of the Set2 SRI domain from the yeast S. cerevisiae and present NMR binding experiments with phospho-CTD peptides. Our results elucidate the molecular determinants for Set2 CTD binding, which underlies coupling of transcription to Set2-directed chromatin modification. Sample Preparation—The region of the gene of the Saccharomyces cerevisiae Set2 protein (Swiss Prot P46995) encoding for Set2 residues 620–719 was cloned into a modified pET9d vector with an NH2-terminal hexahistidine tag. The protein was overexpressed in Escherichia coli pLysS cells at 18 °C for 16 h. For labeling of the protein with 15N/13C or 15N, cells were grown in M9 minimal medium supplemented with [13C6]glucose and/or 15NH4Cl. Cell lysates were subjected to affinity chromatography on a nickel-nitrilotriacetic acid column (Quiagen), followed by cleavage of the hexahistidine tag with tobacco etch virus protease and dialysis overnight. The tag and the His6-tagged protease were removed on a second Ni-NTA column. DNA was removed by cation exchange chromatography (Mono S, Amersham Biosciences). After gel filtration the sample was dissolved in 20 mm sodium phosphate, pH 6.5, 200 mm NaCl, 0.2 mm dithiothreitol. Edman sequencing of the protein confirmed the presence of four additional residues (GAMG) at the NH2 terminus, which result from the cloning strategy. NMR samples were prepared in H2O or 100% D2O at 0.4–1 mm concentration of protein. NMR Structure Determination—NMR spectra were acquired at 292 K on Bruker DRX500, DRX600, or DRX900 spectrometers with cryogenic triple resonance probes. Spectra were processed with NMRPipe (24Delaglio F. Grzesiek S. Vuister G. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed using NMRVIEW (25Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2686) Google Scholar). The 1H, 13C, and 15N chemical shifts were assigned by standard methods (26Sattler M. Schleucher J. Griesinger C. Prog. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1399) Google Scholar). Distance restraints were derived from two-dimensional NOESY and 15N- or 13C-resolved three-dimensional NOESY. Restraints for the backbone angles ϕ and ψ were derived from TALOS (27Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). Slowly exchanging amide protons were identified from 1H,15N correlation experiments after dissolving of lyophilized protein in D2O. 15N relaxation (T1, T2) and heteronuclear (1H)-15N NOE was measured on a 15N-labeled protein sample at 292 K as described (28Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2018) Google Scholar) (supplemental Fig. S1). The experimentally determined distance and dihedral restraints (supplemental Table S1 and Fig. 1C) were applied in a simulated-annealing protocol using ARIA (29Linge J.P. O'Donoghue S.I. Nilges M. Methods Enzymol. 2001; 339: 71-90Crossref PubMed Scopus (333) Google Scholar) and CNS (30Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). NOEs were manually assigned and distance calibrations were performed by ARIA. The final ensemble of NMR structures was refined in a shell of water molecules (31Linge J.P. Williams M.A. Spronk C.A. Bonvin A.M. Nilges M. Proteins. 2003; 50: 496-506Crossref PubMed Scopus (544) Google Scholar). Structural quality was analyzed with PROCHECK (32Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4474) Google Scholar). Phosphopeptide Interaction Studies—The phospho-CTD peptides used for binding experiments were chemically synthesized (one-repeat peptide, YpSPTp-SPS; two-repeat peptide, SPS-YpSPTpSPS-YpSPTpSPS, pS = phosphoserine). For NMR titration, increasing amounts of the CTD peptide were added to a 0.4 mm solution of 15N,13C-labeled SRI domain up to a 1.25-fold molar excess. Chemical shifts were monitored in two-dimensional 1H,15N HSQC experiments. The Set2 SRI Domain Forms a Conserved Three-helix Bundle—The solution structure of the yeast Set2 SRI domain was determined by multidimensional NMR (supplemental Table S1; also see "Experimental Procedures"). The structure revealed three α-helices arranged in a left-handed bundle (Fig. 1). The NH2-terminal helix α1 is slightly kinked at residues Phe639 and Val640, and the linker between helices α1 and α2 includes a short 310-helical turn at residues Ser650– Gln652. A hydrophobic core is formed by numerous residues located at the interface between the three helices, including four residues in the two regions linking the helices (Fig. 1C). Consistently the heteronuclear {1H}-15N NOE measurements demonstrate that the polypeptide backbone in all three helices and the connecting linker regions is rigid (Fig. 1C and supplemental Fig. S1). The hydrophobic core residues are generally conserved across species (Fig. 1C), demonstrating that our structure is a good model for SRI domains in Set2 of other species. The SRI Domain Defines a Novel CTD-binding Fold—Comparison with the five known structures of CTD-binding domains reveals that the SRI domain defines a novel CTD-binding fold. Other CTD-binding domains include FF domains, CTD-interacting domains, WW domains, BRCT domains, and a domain in the Cgt1 subunit of the 5′-capping enzyme (reviewed in Ref. 7Meinhart A. Kamenski T. Hoeppner S. Baumli S. Cramer P. Genes Dev. 2005; 19: 1401-1415Crossref PubMed Scopus (261) Google Scholar). Of these, FF and CTD-interacting domains also form helical bundles (33Allen M. Friedler A. Schon O. Bycroft M. J. Mol. Biol. 2002; 323: 411-416Crossref PubMed Scopus (71) Google Scholar, 34Meinhart A. Cramer P. Nature. 2004; 430: 223-226Crossref PubMed Scopus (232) Google Scholar), but, in contrast to the SRI domain, the superhelical arrangement in these two domains is right-handed (supplemental Fig. S2). Thus the six CTD-binding domains that have been structurally characterized use different folds for specific CTD recognition. The SRI Domain Binds a Two-repeat CTD Phosphopeptide—To characterize the CTD-binding determinants of the SRI domain, we performed NMR titration experiments with Ser2/Ser5-phosphorylated CTD peptides (Fig. 1C). A phosphopeptide consisting of a single CTD repeat (YpSPTpSPS, pS = phosphoserine; Fig. S3A) did not perturb chemical shifts in a two-dimensional 1H,15N HSQC spectrum, indicating that there is no significant binding (data not shown). However, titration with a peptide that comprised two CTD repeats and three flanking NH2-terminal residues (SPS-YpSPTpSPS-YpSPTp-SPS) resulted in many strong chemical shift perturbations (Fig. 1C and supplemental Fig. S3). From the titration data the dissociation constant is estimated to be in the low micromolar range, comparable with the reported approximate affinity of 6 μm for a CTD phosphopeptide comprising three repeats (22Kizer K.O. Phatnani H.P. Shibata Y. Hall H. Greenleaf A.L. Strahl B.D. Mol. Cell Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (367) Google Scholar). Regions in the SRI Domain That Interact with the CTD—Residues that show strong chemical shift perturbations of their backbone NH groups cluster in two regions on the SRI domain structure (Fig. 2A). The first region includes residues Lys634, Phe635 in α1, and Ala662, Val666, Lys667, Thr670, Thr671, and Glu673 in α2, whereas the second region includes residues Phe653, His655, Glu656 in the α1-α2 linker, and residue Ile705 in α3 (Figs. 1C and 2A and supplemental Fig. S3). With the exception of Ile705, the strongest perturbations upon peptide binding were observed in region 1 (Phe635, Ala662, Val666, Lys667, and Glu673). In this region, the side chain NH2 groups of residues Asn631 and Asn633 also show significant chemical shift perturbations (supplemental Fig. S3B). Both regions are conserved among fungal Set2 homologues (Fig. 2B), befitting the conserved function of the Saccharomyces pombe and Neurospora crassa Set2 homologues (35Morris S.A. Shibata Y. Noma K. Tsukamoto Y. Warren E. Temple B. Grewal S.I. Strahl B.D. Eukaryot. Cell. 2005; 4: 1446-1454Crossref PubMed Scopus (90) Google Scholar, 36Adhvaryu K.K. Morris S.A. Strahl B.D. Selker E.U. Eukaryot. Cell. 2005; 4: 1455-1464Crossref PubMed Scopus (70) Google Scholar). The observation of two putative CTD-binding regions, and the finding that two CTD repeats are required for SRI domain binding, indicate that the phospho-CTD extends over a long distance along helices α1 and α2 and the connecting linker. CTD Tyrosine Side Chains Contribute to SRI Domain Binding—The peptide titration experiments also revealed that the two-repeat CTD peptide (supplemental Fig. S3A) binds to the SRI domain via its tyrosine residues. Intermolecular NOEs between both CTD tyrosine side chains and the SRI domain were detected (data not shown). Preliminary assignments indicate that one of the tyrosine side chains is in proximity of residues Ala662 and Val666 in region 1 (Figs. 1C and 2B). These two residues are part of a hydrophobic patch between helices α1 and α2 and flanked by positively charged surfaces (Fig. 2C), as expected for interaction with the negatively charged phospho-CTD. Interestingly, the tyrosine-proximal residue Ala662 is identical in human Set2, as are Phe635, Glu656, and Glu673 in the putative CTD-binding regions (Fig. 1C). In the three known CTD-protein complex structures, the Y1 side chain is also involved in hydrophobic contacts (34Meinhart A. Cramer P. Nature. 2004; 430: 223-226Crossref PubMed Scopus (232) Google Scholar, 37Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 38Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (428) Google Scholar), suggesting that Y1 binding is a general feature of CTD recognition. Previous studies revealed that the CTD can adopt different conformations (reviewed in Ref. 7Meinhart A. Kamenski T. Hoeppner S. Baumli S. Cramer P. Genes Dev. 2005; 19: 1401-1415Crossref PubMed Scopus (261) Google Scholar), and this structurally versatile nature of the CTD discourages any detailed model building. The SRI Domain Resembles a Polymerase-interacting Domain in Bacterial σ Factors—Comparison of our structure with known folds in the data base (DALI (39Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1291) Google Scholar)) strikingly shows that the SRI domain resembles a region in bacterial σ factors (Fig. 3). The four highest hits were the σ factors σ28 (PDB code 1rp3), σE (PDB-code 1or7), σR (PDB code 1h3l), and σ70 (PDB code 1sig), which show DALI scores of 5.6, 5.4, 5.1, and 4.9, respectively, and root mean square deviations between 3.3 and 3.7 Å. The region in σ70 that is structurally related to the SRI domain is domain 2 (σ2), which interacts with the clamp region of the core RNA polymerase upon formation of the holoenzyme (40Murakami K.S. Masuda S. Darst S.A. Science. 2002; 296: 1280-1284Crossref PubMed Scopus (449) Google Scholar). The σ2 domain is involved in binding the –10 element of promoter DNA and contributes to DNA melting during initiation (reviewed in Ref. 41Gross C.A. Chan C. Dombroski A. Gruber T. Sharp M. Tupy J. Young B. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 141-155Crossref PubMed Scopus (298) Google Scholar). In the eukaryotic initiation complex, promoter DNA around position –10 lies near the NH2-terminal domain of the initiation factor TFIIEα (42Forget D. Langelier M.-F. Therien C. Trinh V. Coulombe B. Mol. Cell. Biol. 2004; 24: 1122-1131Crossref PubMed Scopus (53) Google Scholar), which shows weak sequence homology (43Okhuma Y. Sumimoto H. Hoffmann A. Shimasaki S. Horikoshhi M. Roeder R. Nature. 1991; 354: 398-401Crossref PubMed Scopus (73) Google Scholar) and structural similarity (44Meinhart A. Blobel J. Cramer P. J. Biol. Chem. 2003; 278: 48267-48274Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) to the bacterial σ2 domain. We speculate that the eukaryotic TFIIEα NH2-terminal domain, which may contact promoter DNA, and the Set2 SRI domain, which binds the negatively charged phospho-CTD, both evolved from the bacterial σ2 domain. We thank C. Buchen, L. Lariviere, and other members of the Cramer laboratory (Gene Center Munich) for help. We thank G. Stier and A. Lingel (EMBL, Heidelberg, Germany) and K. Kizer (University of North Carolina) for help. Download .pdf (1.02 MB) Help with pdf files
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