Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code.

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

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Meinhart A, Kamenski T, Hoeppner S, Baumli S, Cramer P (2005) A structural perspective of CTD function. Genes Dev 19: 1401–1415 PubMed

Jasnovidova O, Stefl R (2013) The CTD code of RNA polymerase II: a structural view. Wiley Interdiscip Rev RNA 4: 1–16 PubMed

Eick D, Geyer M (2013) The RNA polymerase II carboxy‐terminal domain (CTD) code. Chem Rev 113: 8456–8490 PubMed

Zaborowska J, Egloff S, Murphy S (2016) The pol II CTD: new twists in the tail. Nat Struct Mol Biol 23: 771–777 PubMed

Jeronimo C, Collin P, Robert F (2016) The RNA polymerase II CTD: the increasing complexity of a low‐complexity protein domain. J Mol Biol 428: 2607–2622 PubMed

Chapman RD, Heidemann M, Hintermair C, Eick D (2008) Molecular evolution of the RNA polymerase II CTD. Trends Genet 24: 289–296 PubMed

Jeronimo C, Bataille AR, Robert F (2013) The writers, readers, and functions of the RNA polymerase ii c‐terminal domain code. Chem Rev 113: 8491–8522 PubMed

Sakurai H, Ishihama A (2002) Level of the RNA polymerase II in the fission yeast stays constant but phosphorylation of its carboxyl terminal domain varies depending on the phase and rate of cell growth. Genes Cells 7: 273–284 PubMed

Rosonina E, Yurko N, Li W, Hoque M, Tian B, Manley JL (2014) Threonine‐4 of the budding yeast RNAP II CTD couples transcription with Htz1‐mediated chromatin remodeling. Proc Natl Acad Sci 111: 11924–11931 PubMed PMC

Schüller R, Forné I, Straub T, Schreieck A, Texier Y, Shah N, Decker T‐M, Cramer P, Imhof A, Eick D (2016) Heptad‐specific phosphorylation of RNA polymerase II CTD. Mol Cell 61: 305–314 PubMed

Suh H, Ficarro SB, Kang U‐B, Chun Y, Marto JA, Buratowski S (2016) Direct analysis of phosphorylation sites on the Rpb1 C‐terminal domain of RNA polymerase II. Mol Cell 61: 297–304 PubMed PMC

Hintermair C, Heidemann M, Koch F, Descostes N, Gut M, Gut I, Fenouil R, Ferrier P, Flatley A, Kremmer E et al (2012) Threonine‐4 of mammalian RNA polymerase II CTD is targeted by Polo‐like kinase 3 and required for transcriptional elongation. EMBO J 31: 2784–2797 PubMed PMC

Hsin J‐P, Sheth A, Manley JL (2011) RNAP II CTD phosphorylated on Threonine‐4 is required for histone mRNA 3′ end processing. Science 334: 683–686 PubMed PMC

Hintermair C, Voß K, Forné I, Heidemann M, Flatley A, Kremmer E, Imhof A, Eick D (2016) Specific threonine‐4 phosphorylation and function of RNA polymerase II CTD during M phase progression. Sci Rep 6: 27401 PubMed PMC

Stiller JW, McConaughy BL, Hall BD (2000) Evolutionary complementation for polymerase II CTD function. Yeast 16: 57–64 PubMed

Schwer B, Shuman S (2011) Deciphering the RNA polymerase II CTD code in fission yeast. Mol Cell 43: 311–318 PubMed PMC

Mayer A, Heidemann M, Lidschreiber M, Schreieck A, Sun M, Hintermair C, Kremmer E, Eick D, Cramer P (2012) CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II. Science 336: 1723–1725 PubMed

Harlen KM, Trotta KL, Smith EE, Mosaheb MM, Fuchs SM, Churchman LS (2016) Comprehensive RNA polymerase II interactomes reveal distinct and varied roles for each phospho‐CTD residue. Cell Rep 15: 2147–2158 PubMed PMC

Kim M, Krogan NJ, Vasiljeva L, Rando OJ, Nedea E, Greenblatt JF, Buratowski S (2004) The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432: 517–522 PubMed

Lunde BM, Reichow SL, Kim M, Suh H, Leeper TC, Yang F, Mutschler H, Buratowski S, Meinhart A, Varani G (2010) Cooperative interaction of transcription termination factors with the RNA polymerase II C‐terminal domain. Nat Struct Mol Biol 17: 1195–1201 PubMed PMC

Ni Z, Xu C, Guo X, Hunter GO, Kuznetsova OV, Tempel W, Marcon E, Zhong G, Guo H, Kuo W‐HW et al (2014) RPRD1A and RPRD1B are human RNA polymerase II C‐terminal domain scaffolds for Ser5 dephosphorylation. Nat Struct Mol Biol 21: 686–695 PubMed PMC

Becker R, Loll B, Meinhart A (2008) Snapshots of the RNA processing factor SCAF8 bound to different phosphorylated forms of the carboxyl‐terminal domain of RNA polymerase II. J Biol Chem 283: 22659–22669 PubMed

Kubicek K, Cerna H, Holub P, Pasulka J, Hrossova D, Loehr F, Hofr C, Vanacova S, Stefl R (2012) Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1. Genes Dev 26: 1891–1896 PubMed PMC

Xiang K, Nagaike T, Xiang S, Kilic T, Beh MM, Manley JL, Tong L (2010) Crystal structure of the human symplekin‐Ssu72‐CTD phosphopeptide complex. Nature 467: 729–733 PubMed PMC

Werner‐Allen JW, Lee C‐J, Liu P, Nicely NI, Wang S, Greenleaf AL, Zhou P (2011) cis‐Proline‐mediated Ser(P)5 dephosphorylation by the RNA polymerase II C‐terminal domain phosphatase Ssu72. J Biol Chem 286: 5717–5726 PubMed PMC

Meinhart A, Cramer P (2004) Recognition of RNA polymerase II carboxy‐terminal domain by 3’‐RNA‐processing factors. Nature 430: 223–226 PubMed

Noble CG, Hollingworth D, Martin SR, Ennis‐Adeniran V, Smerdon SJ, Kelly G, Taylor IA, Ramos A (2005) Key features of the interaction between Pcf11 CID and RNA polymerase II CTD. Nat Struct Mol Biol 12: 144–151 PubMed

Buratowski S (2003) The CTD code. Nat Struct Mol Biol 10: 679–680 PubMed

Kay LE, Xu GY, Singer AU, Muhandiram DR, Forman‐Kay JD (1993) A gradient‐enhanced HCCH TOCSY experiment for recording side‐chain H‐1 and C‐13 correlations in H2O samples of proteins. J Magn Reson Ser 101: 333–337

Nováček J, Haba NY, Chill JH, Žídek L, Sklenář V (2012) 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 53: 139–148 PubMed

Nováček J, Zawadzka‐Kazimierczuk A, Papoušková V, Žídek L, Šanderová H, Krásný L, Koźmiński W, Sklenář V (2011) 5D 13C‐detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion. J Biomol NMR 50: 1–11 PubMed

Peterson RD, Theimer CA, Wu H, Feigon J (2004) New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA‐protein complexes. J Biomol NMR 28: 59–67 PubMed

Zwahlen C, Legault P, Vincent SJF, Greenblatt J, Konrat R, Kay LE (1997) Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: application to a bacteriophage λ N‐Peptide/boxB RNA complex. J Am Chem Soc 119: 6711–6721

Güntert P, Buchner L (2015) Combined automated NOE assignment and structure calculation with CYANA. J Biomol NMR 62: 453–471 PubMed

Case DA, Betz RM, Botello‐Smith W, Cerutti DS, Cheatham TE, Darden TA, Duke RE, Giese TJ (2016) AMBER 2016. San Francisco: University of California;

Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C (2015) ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput 11: 3696–3713 PubMed PMC

Stefl R, Oberstrass FC, Hood JL, Jourdan M, Zimmermann M, Skrisovska L, Maris C, Peng L, Hofr C, Emeson RB et al (2010) The solution structure of the ADAR2 dsRBM‐RNA complex reveals a sequence‐specific readout of the minor groove. Cell 143: 225–237 PubMed PMC

Hobor F, Pergoli R, Kubicek K, Hrossova D, Bacikova V, Zimmermann M, Pasulka J, Hofr C, Vanacova S, Stefl R (2011) Recognition of transcription termination signal by the nuclear polyadenylated RNA‐binding (NAB) 3 protein. J Biol Chem 286: 3645–3657 PubMed PMC

Kuzmic P (2009) DynaFit–a software package for enzymology. Methods Enzymol 467: 247–280 PubMed

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612 PubMed

Schubert M, Labudde D, Oschkinat H, Schmieder P (2002) A software tool for the prediction of Xaa‐Pro peptide bond conformations in proteins based on 13C chemical shift statistics. J Biomol NMR 24: 149–154 PubMed

Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK‐NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8: 477–486 PubMed

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