Transcription elongation factor Spt6 associates with RNA polymerase II (RNAP II) via a tandem SH2 (tSH2) domain. The mechanism and significance of the RNAP II-Spt6 interaction is still unclear. Recently, it was proposed that Spt6-tSH2 is recruited via a newly described phosphorylated linker between the Rpb1 core and its C-terminal domain (CTD). Here, we report binding studies with isolated tSH2 of Spt6 (Spt6-tSH2) and Spt6 lacking the first unstructured 297 residues (Spt6ΔN) with a minimal CTD substrate of two repetitive heptads phosphorylated at different sites. The data demonstrate that Spt6 also binds the phosphorylated CTD, a site that was originally proposed as a recognition epitope. We also show that an extended CTD substrate harboring 13 repetitive heptads of the tyrosine-phosphorylated CTD binds Spt6-tSH2 and Spt6ΔN with tighter affinity than the minimal CTD substrate. The enhanced binding is achieved by avidity originating from multiple phosphorylation marks present in the CTD. Interestingly, we found that the steric effects of additional domains in the Spt6ΔN construct partially obscure the binding of the tSH2 domain to the multivalent ligand. We show that Spt6-tSH2 binds various phosphorylation patterns in the CTD and found that the studied combinations of phospho-CTD marks (1,2; 1,5; 2,4; and 2,7) all facilitate the interaction of CTD with Spt6. Our structural studies reveal a plasticity of the tSH2 binding pockets that enables the accommodation of CTDs with phosphorylation marks in different registers.

CEITEC Central European Institute of Technology Masaryk University CZ 62500 Brno Czech Republic

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Corden J.L., Cadena D.L., Ahearn J.M., Dahmus M.E. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc. Natl. Acad. Sci. 1985;82:7934–7938. doi: 10.1073/pnas.82.23.7934. PubMed DOI PMC

Buratowski S. The CTD code. Nat. Struct. Mol. Biol. 2003;10:679–680. doi: 10.1038/nsb0903-679. PubMed DOI

Egloff S., Murphy S. Cracking the RNA polymerase II CTD code. Trends Genet. 2008;24:280–288. doi: 10.1016/j.tig.2008.03.008. PubMed DOI

Eick D., Geyer M. The RNA polymerase II carboxy-terminal domain (CTD) code. Chem. Rev. 2013;113:8456–8490. doi: 10.1021/cr400071f. PubMed DOI

Cho W.-K., Spille J.-H., Hecht M., Lee C., Li C., Grube V., Cisse I.I. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science. 2018;361:412–415. doi: 10.1126/science.aar4199. PubMed DOI PMC

Boehning M., Dugast-Darzacq C., Rankovic M., Hansen A.S., Yu T., Marie-Nelly H., McSwiggen D.T., Kokic G. RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat. Struct. Mol. Biol. 2018;25:833–840. doi: 10.1038/s41594-018-0112-y. PubMed DOI

Lu H., Yu D., Hansen A.S., Ganguly S., Liu R., Heckert A., Darzacq X., Zhou Q. Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature. 2018;558:318–323. doi: 10.1038/s41586-018-0174-3. PubMed DOI PMC

Corden J.L. RNA polymerase II C-terminal domain: tethering transcription to transcript and template. Chem. Rev. 2013;113:8423–8455. doi: 10.1021/cr400158h. PubMed DOI PMC

Mayer A., Heidemann M., Lidschreiber M., Schreieck A., Sun M., Hintermair C., Kremmer E., Eick D. CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II. Science. 2012;336:1723–1725. doi: 10.1126/science.1219651. PubMed DOI

Swanson M.S., Carlson M., Winston F. SPT6, an essential gene that affects transcription in Saccharomyces cerevisiae, encodes a nuclear protein with an extremely acidic amino terminus. Mol. Cell. Biol. 1990;10:4935–4941. doi: 10.1128/MCB.10.9.4935. PubMed DOI PMC

Kaplan C.D., Moris J.R., Wu C., Winston F. Spt5 and Spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 2000;14:2623–2634. doi: 10.1101/gad.831900. PubMed DOI PMC

Perales R., Erickson B., Zhang L., Kim H., Valiquett E., Bentley D. Gene promoters dictate histone occupancy within genes. EMBO J. 2013;32:2645–2656. doi: 10.1038/emboj.2013.194. PubMed DOI PMC

McDonald S.M., Close D., Xin H., Formosa T., Hill C.P. Structure and biological importance of the Spn1–Spt6 interaction, and its regulatory role in nucleosome binding. Mol. Cell. 2010;40:725–735. doi: 10.1016/j.molcel.2010.11.014. PubMed DOI PMC

McCullough L., Connell Z., Petersen C., Formosa T. The abundant histone chaperones Spt6 and FACT collaborate to assemble, inspect, and maintain chromatin structure in Saccharomyces cerevisiae. Genetics. 2015;201:1031–1045. doi: 10.1534/genetics.115.180794. PubMed DOI PMC

Kaplan C.D., Laprade L., Winston F. Transcription elongation factors repress transcription initiation from cryptic sites. Science. 2003;301:1096–1099. doi: 10.1126/science.1087374. PubMed DOI

DeGennaro C.M., Alver B.H., Marguerat S., Stepanova E., Davis C.P., Bahler J., Park P.J., Winston F. Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast. Mol. Cell. Biol. 2013;33:4779–4792. doi: 10.1128/MCB.01068-13. PubMed DOI PMC

Doris S.M., Chuang J., Viktorovskaya O., Murawska M., Spatt D., Churchman L.S., Winston F. Spt6 is required for the fidelity of promoter selection. BioRxiv. 2018 doi: 10.1101/347575. PubMed DOI PMC

Kato H., Okazaki K., Iida T., Nakayama J., Murakami Y., Urano T. Spt6 prevents transcription-coupled loss of posttranslationally modified histone H3. Sci. Rep. 2013;3 doi: 10.1038/srep02186. PubMed DOI PMC

Carrozza M.J., Li B., Florens L., Suganuma T., Swanson S.K., Lee K.K., Shia W.-J., Anderson S. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell. 2005;123:581–592. doi: 10.1016/j.cell.2005.10.023. PubMed DOI

Mayer A., Lidschreiber M., Siebert M., Leike K., Söding J., Cramer P. Uniform transitions of the general RNA polymerase II transcription complex. Nat. Struct. Mol. Biol. 2010;17:1272–1278. doi: 10.1038/nsmb.1903. PubMed DOI

Sdano M.A., Fulcher J.M., Palani S., Chandrasekharan M.B., Parnell T.J., Whitby F.G., Formosa T., Hill C.P. A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription. ELife. 2017;6 doi: 10.7554/eLife.28723. PubMed DOI PMC

Belotserkovskaya R., Reinberg D. Facts about FACT and transcript elongation through chromatin. Curr. Opin. Genet. Dev. 2004;14:139–146. doi: 10.1016/j.gde.2004.02.004. PubMed DOI

Adkins M.W., Tyler J.K. Transcriptional activators are dispensable for transcription in the absence of Spt6-mediated chromatin reassembly of promoter regions. Mol. Cell. 2006;21:405–416. doi: 10.1016/j.molcel.2005.12.010. PubMed DOI

Nojima T., Tellier M., Foxwell J., Ribeiro de Almeida C., Tan-Wong S.M., Dhir S., Dujardin G., Dhir A. Deregulated expression of mammalian lncRNA through loss of SPT6 induces R-loop formation, replication stress, and cellular senescence. Mol. Cell. 2018;72:970–984.e7. doi: 10.1016/j.molcel.2018.10.011. PubMed DOI PMC

Endoh M., Zhu W., Hasegawa J., Watanabe H., Kim D.-K., Aida M., Inukai N., Narita T. Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. Mol. Cell. Biol. 2004;24:3324–3336. doi: 10.1128/MCB.24.8.3324-3336.2004. PubMed DOI PMC

Ardehali M.B., Yao J., Adelman K., Fuda N.J., Petesch S.J., Webb W.W., Lis J.T. Spt6 enhances the elongation rate of RNA polymerase II in vivo. EMBO J. 2009;28:1067–1077. doi: 10.1038/emboj.2009.56. PubMed DOI PMC

Kaplan C.D., Holland M.J., Winston F. Interaction between transcription elongation factors and mRNA 3′-end formation at the Saccharomyces cerevisiae GAL10–GAL7 locus. J. Biol. Chem. 2005;280:913–922. doi: 10.1074/jbc.M411108200. PubMed DOI

Yoh S.M., Cho H., Pickle L., Evans R.M., Jones K.A. The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev. 2007;21:160–174. doi: 10.1101/gad.1503107. PubMed DOI PMC

Diebold M.-L., Loeliger E., Koch M., Winston F., Cavarelli J., Romier C. Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II. J. Biol. Chem. 2010;285:38389–38398. doi: 10.1074/jbc.M110.146696. PubMed DOI PMC

Sun M., Larivière L., Dengl S., Mayer A., Cramer P. A tandem SH2 domain in transcription elongation factor Spt6 binds the phosphorylated RNA polymerase II C-terminal repeat domain (CTD) J. Biol. Chem. 2010;285:41597–41603. doi: 10.1074/jbc.M110.144568. PubMed DOI PMC

Close D., Johnson S.J., Sdano M.A., McDonald S.M., Robinson H., Formosa T., Hill C.P. Crystal structures of the S. cerevisiae Spt6 core and C-terminal tandem SH2 domain. J. Mol. Biol. 2011;408:697–713. doi: 10.1016/j.jmb.2011.03.002. PubMed DOI PMC

Vos S.M., Farnung L., Boehning M., Wigge C., Linden A., Urlaub H., Cramer P. Structure of activated transcription complex Pol II–DSIF–PAF–SPT6. Nature. 2018;560:607–612. doi: 10.1038/s41586-018-0440-4. PubMed DOI

Dronamraju R., Hepperla A.J., Shibata Y., Adams A.T., Magnuson T., Davis I.J., Strahl B.D. Spt6 association with RNA polymerase II directs mRNA turnover during transcription. Mol. Cell. 2018;70:1054–1066.e4. doi: 10.1016/j.molcel.2018.05.020. PubMed DOI PMC

Tripsianes K., Chu N.K., Friberg A., Sattler M., Becker C.F.W. Studying weak and dynamic interactions of posttranslationally modified proteins using expressed protein ligation. ACS Chem. Biol. 2014;9:347–352. doi: 10.1021/cb400723j. PubMed DOI

Tripsianes K., Madl T., Machyna M., Fessas D., Englbrecht C., Fischer U., Neugebauer K.M., Sattler M. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nat. Struct. Mol. Biol. 2011;18:1414–1420. doi: 10.1038/nsmb.2185. PubMed DOI

Brazda P., Sedo O., Kubicek K., Stefl R. Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins. Biotechniques. 2019;66 PubMed

Jasnovidova O., Stefl R. The CTD code of RNA polymerase II: a structural view: the CTD code of RNA polymerase II. Wiley Interdiscip. Rev. RNA. 2013;4:1–16. doi: 10.1002/wrna.1138. PubMed DOI

Jasnovidova O., Krejcikova M., Kubicek K., Stefl R. Structural insight into recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by Rtt103p. EMBO Rep. 2017;18:906–913. doi: 10.15252/embr.201643723. PubMed DOI PMC

Heyduk T., Lee J.C. Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction. Proc. Natl. Acad. Sci. 1990;87:1744–1748. doi: 10.1073/pnas.87.5.1744. PubMed DOI PMC

Tudek A., Porrua O., Kabzinski T., Lidschreiber M., Kubicek K., Fortova A., Lacroute F., Vanacova S. Molecular basis for coordinating transcription termination with noncoding RNA degradation. Mol. Cell. 2014;55:467–481. doi: 10.1016/j.molcel.2014.05.031. PubMed DOI PMC

Pasquier C., Vazdar M., Forsman J., Jungwirth P., Lund M. Anomalous protein–protein interactions in multivalent salt solution. J. Phys. Chem. B. 2017;121:3000–3006. doi: 10.1021/acs.jpcb.7b01051. PubMed DOI

Li W., Persson B.A., Lund M., Bergenholtz J., Zackrisson Oskolkova M. Concentration-induced association in a protein system caused by a highly directional patch attraction. J. Phys. Chem. B. 2016;120:8953–8959. doi: 10.1021/acs.jpcb.6b06873. PubMed DOI

McWilliam H., Li W., Uludag M., Squizzato S., Park Y.M., Buso N., Cowley A.P., Lopez R. Analysis tool web services from the EMBL-EBI. Nucleic Acids Res. 2013;41:W597–W600. doi: 10.1093/nar/gkt376. PubMed DOI PMC

Robert X., Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014;42:W320–W324. doi: 10.1093/nar/gku316. PubMed DOI PMC

Waksman G., Shoelson S.E., Pant N., Cowburn D., Kuriyan J. Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms. Cell. 1993;72:779–790. PubMed

Narula S., Yuan R., Adams S., Green O., Green J., Philips T., Zydowsky L., Botfield M. Solution structure of the C-terminal SH2 domain of the human tyrosine kinase Syk complexed with a phosphotyrosine pentapeptide. Structure. 1995;3:1061–1073. doi: 10.1016/S0969-2126(01)00242-8. PubMed DOI

Spahr H., Calero G., Bushnell D.A., Kornberg R.D. Schizosacharomyces pombe RNA polymerase II at 3.6-A resolution. Proc. Natl. Acad. Sci. 2009;106:9185–9190. doi: 10.1073/pnas.0903361106. PubMed DOI PMC

Suh H., Hazelbaker D.Z., Soares L.M., Buratowski S. The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits. Mol. Cell. 2013;51:850–858. doi: 10.1016/j.molcel.2013.08.015. PubMed DOI PMC

Mourão A., Bonnal S., Soni K., Warner L., Bordonné R., Valcárcel J., Sattler M. Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation. ELife. 2016;5 doi: 10.7554/eLife.14707. PubMed DOI PMC

Hlavacek W.S., Posner R.G., Perelson A.S. Steric effects on multivalent ligand-receptor binding: exclusion of ligand sites by bound cell surface receptors. Biophys. J. 1999;76:3031–3043. doi: 10.1016/S0006-3495(99)77456-4. PubMed DOI PMC

Fabrega C., Shen V., Shuman S., Lima C.D. Structure of an mRNA capping enzyme bound to the phosphorylated carboxy-terminal domain of RNA polymerase II. Mol. Cell. 2003;11:1549–1561. doi: 10.1016/S1097-2765(03)00187-4. PubMed DOI

Kubicek K., Cerna H., Holub P., Pasulka J., Hrossova D., Loehr F., Hofr C., Vanacova S. Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1. Genes Dev. 2012;26:1891–1896. doi: 10.1101/gad.192781.112. PubMed DOI PMC

Lunde B.M., Reichow S.L., Kim M., Suh H., Leeper T.C., Yang F., Mutschler H., Buratowski S. Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nat. Struct. Mol. Biol. 2010;17:1195–1201. doi: 10.1038/nsmb.1893. PubMed DOI PMC

Harlen K.M., Trotta K.L., Smith E.E., Mosaheb M.M., Fuchs S.M., Churchman L.S. Comprehensive RNA polymerase II interactomes reveal distinct and varied roles for each phospho-CTD residue. Cell Rep. 2016;15:2147–2158. doi: 10.1016/j.celrep.2016.05.010. PubMed DOI PMC

Harlen K.M., Churchman L.S. Subgenic Pol II interactomes identify region-specific transcription elongation regulators. Mol. Syst. Biol. 2017;13:900. doi: 10.15252/msb.20167279. PubMed DOI PMC

Schüller R., Forné I., Straub T., Schreieck A., Texier Y., Shah N., Decker T.-M., Cramer P. Heptad-specific phosphorylation of RNA polymerase II CTD. Mol. Cell. 2016;61:305–314. doi: 10.1016/j.molcel.2015.12.003. PubMed DOI

Suh H., Ficarro S.B., Kang U.-B., Chun Y., Marto J.A., Buratowski S. Direct analysis of phosphorylation sites on the Rpb1 C-terminal domain of RNA polymerase II. Mol. Cell. 2016;61:297–304. doi: 10.1016/j.molcel.2015.12.021. PubMed DOI PMC

Sattler M., Schleucher J., Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 1999;34:93–158. doi: 10.1016/S0079-6565(98)00025-9. DOI

Kay L.E., Xu G.Y., Singer A.U., Muhandiram D.R., Formankay J.D. A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 1H and 13C correlations in H 2O samples of proteins. J. Magn. Reson. 1993;101:333–337. doi: 10.1006/jmrb.1993.1053. DOI

Hoch J.C., Maciejewski M.W., Mobli M., Schuyler A.D., Stern A.S. Nonuniform sampling and maximum entropy reconstruction in multidimensional NMR. Acc. Chem. Res. 2014;47:708–717. doi: 10.1021/ar400244v. PubMed DOI PMC

Nováček J., Haba N.Y., Chill J.H., Žídek L., Sklenář V. 4D non-uniformly sampled HCBCACON and 1J(NCα)-selective HCBCANCO experiments for the sequential assignment and chemical shift analysis of intrinsically disordered proteins. J. Biomol. NMR. 2012;53:139–148. doi: 10.1007/s10858-012-9631-8. PubMed DOI

Nováček J., Zawadzka-Kazimierczuk A., Papoušková V., Žídek L., Šanderová H., Krásný L., Koźmiński W., Sklenář V. 5D 13C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion. J. Biomol. NMR. 2011;50:1–11. doi: 10.1007/s10858-011-9496-2. PubMed DOI

Peterson R.D., Theimer C.A., Wu H., Feigon J. New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA–protein complexes. J. Biomol. NMR. 2004;28:59–67. doi: 10.1023/B:JNMR.0000012861.95939.05. PubMed DOI

Zwahlen C., Legault P., Vincent S.J.F., Greenblatt J., Konrat R., Kay L.E. Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: application to a bacteriophage λ N-peptide/boxB RNA complex. J. Am. Chem. Soc. 1997;119:6711–6721. doi: 10.1021/ja970224q. DOI

Güntert P., Buchner L. Combined automated NOE assignment and structure calculation with CYANA. J. Biomol. NMR. 2015;62:453–471. doi: 10.1007/s10858-015-9924-9. PubMed DOI

Case D.A., Betz R.M., Cerutti D.S., Cheatham T., Darden T., Duke R.E., Giese T.J., Gohlke H. Univ. Calif. San Franc; 2016. Amber 16, University of California, San Francisco. DOI

Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E., Simmerling C. ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015;11:3696–3713. doi: 10.1021/acs.jctc.5b00255. PubMed DOI PMC

Homeyer N., Horn A.H.C., Lanig H., Sticht H. AMBER force-field parameters for phosphorylated amino acids in different protonation states: phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine. J. Mol. Model. 2006;12:281–289. doi: 10.1007/s00894-005-0028-4. PubMed DOI

Hobor F., Pergoli R., Kubicek K., Hrossova D., Bacikova V., Zimmermann M., Pasulka J., Hofr C. Recognition of transcription termination signal by the nuclear polyadenylated RNA-binding (NAB) 3 protein. J. Biol. Chem. 2011;286:3645–3657. doi: 10.1074/jbc.M110.158774. PubMed DOI PMC

Stefl R., Oberstrass F.C., Hood J.L., Jourdan M., Zimmermann M., Skrisovska L., Maris C., Peng L. The solution structure of the ADAR2 dsRBM–RNA complex reveals a sequence-specific readout of the minor groove. Cell. 2010;143:225–237. doi: 10.1016/j.cell.2010.09.026. PubMed DOI PMC

Battiste J.L., Wagner G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear Overhauser effect data. Biochemistry. 2000;39:5355–5365. doi: 10.1021/bi000060h. PubMed DOI

Roosild T.P., Greenwald J., Vega M., Castronovo S., Riek R., Choe S. NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science. 2005;307:1317–1321. doi: 10.1126/science.1106392. PubMed DOI

Svergun D., Barberato C., Koch M.H.J. CRYSOL—a program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 1995;28:768–773. doi: 10.1107/S0021889895007047. DOI

Stenqvist B., Thuresson A., Kurut A., Vácha R., Lund M. Faunus—a flexible framework for Monte Carlo simulation. Mol. Simul. 2013;39:1233–1239. doi: 10.1080/08927022.2013.828207. DOI

Bagotsky V.S. 2nd ed. Wiley-Interscience; Hoboken, NJ: 2005. Fundamentals of Electrochemistry.

Israelachvili J.N. 3rd ed. Academic Press; 2015. Intermolecular and Surface Forces.

Frenkel D., Smit B. 2d ed. Academic Press; San Diego: 2001. Understanding Molecular Simulation: From Algorithms to Applications.

Abraham M.J., Murtola T., Schulz R., Páll S., Smith J.C., Hess B., Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25. doi: 10.1016/j.softx.2015.06.001. DOI

Lund M., Åkesson T., Jönsson B. Enhanced protein adsorption due to charge regulation. Langmuir. 2005;21:8385–8388. doi: 10.1021/la050607z. PubMed DOI

Hoffmann R., Reichert I., Wachs W.O., Zeppezauer M., Kalbitzer H.R. 1H and 31P NMR spectroscopy of phosphorylated model peptides. Int. J. Pept. Protein Res. 2009;44:193–198. doi: 10.1111/j.1399-3011.1994.tb00160.x. PubMed DOI

Cooperation between intrinsically disordered and ordered regions of Spt6 regulates nucleosome and Pol II CTD binding, and nucleosome assembly