Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain

Journal name:
Nature Structural & Molecular Biology
Year published:
Published online


Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTD-interacting domains (CIDs) that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CIDs of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single-residue mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′ end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Ser2 phosphorylation density is highest.

At a glance


  1. Sequence alignment of CIDs and diheptad CTD phosphopeptides used in this study.
    Figure 1: Sequence alignment of CIDs and diheptad CTD phosphopeptides used in this study.

    (a) Sequence alignment of yeast (Sc) Rtt103, Pcf11, Nrd1 and human (Hs) SCAF8 and SCAF4 CIDs. Red, identical residues; purple, conserved amino acids. Secondary structure of Rtt103 is above the sequence. Blue dots identify Rtt103 residues that bind to the CTD in our structure. Cyan box marks positions of Arg108 and Asn107 in Rtt103 and Pcf11, respectively, and the corresponding residues in the Nrd1 and the SCAF CIDs. (b) Schematic of diheptad CTD peptides used in this study. Sequence at top; sites of serine phosphorylation, orange circles.

  2. Binding of Rtt103-CID and Pcf11-CID to diheptad CTD phosphopeptides monitored by fluorescence anisotropy and NMR.
    Figure 2: Binding of Rtt103-CID and Pcf11-CID to diheptad CTD phosphopeptides monitored by fluorescence anisotropy and NMR.

    (a) Rtt103-CID titrated into 2 μM FAM–labeled Ser2P-CTD diheptad repeat peptide (•), which competes against 25 μM Ser2P-CTD diheptad repeat peptides (), 50 μM Ser5P-CTD diheptad repeat peptides (), and 50 μM Ser2P and Ser5P-CTD diheptad repeat peptides () for binding. (b) Titration of Pcf11, as described in a except that 300 μM of unlabeled peptides were used. (c) Superposition of 1H-15N HSQC spectra of Rtt103 at several points in the titration with the doubly Ser2-phosphorylated CTD peptide showing changes in chemical shift on peptide binding. (d) HSQC perturbations were used to calculate binding affinities on a residue-by-residue basis for all amino acids showing substantial chemical shift changes on peptide binding by plotting the change in chemical shift versus the peptide concentration and by fitting the data to equation (1) in Supplementary Methods.

  3. Structure of Rtt103-CID and recognition of the Ser2P CTD.
    Figure 3: Structure of Rtt103-CID and recognition of the Ser2P CTD.

    (a) Ensemble of the 20 lowest-energy structures of the free Rtt103-CID as determined by NMR. (b) Superposition of the four existing CID structures: Rtt103, green, this study; Pcf11, cyan24; SCAF8, pink22 and Nrd1, gray20. (c) Structure of Rtt103 bound to the Ser2P (S2b-P) CTD (yellow). (d) Close-up of recognition of phosphorylated CTD by Rtt103 highlighting residues that directly contact the peptide. This and all subsequent structural figures were generated with PyMOL36.

  4. CTD specificity can be altered by a single-residue change.
    Figure 4: CTD specificity can be altered by a single-residue change.

    (a) Arg108 in Rtt103 makes several direct contacts to the CTD Ser2 phosphate (S2b-P); it is within hydrogen bonding distance of both the phosphoserine (S2bP) and the threonine (T4b) in the CTD (dotted black lines). Several other hydrogen bonds (dotted black lines) are characteristic of the β-turn structure. (b) Affinities (± s.d.) for wild-type and mutant Rtt103 and Pcf11 CIDs determined by anisotropy and NMR, respectively, with the Ser2abP diheptad CTD peptide. (c) Recruitment of C-terminally TAP-tagged Rtt103 and Rtt103 R108N to the highly transcribed PMA1 was monitored by ChIP assay. Immunoprecipitated DNA was amplified with PMA1 primers as diagrammed at bottom. Top, PMA1-specific; common lower band (star), internal background control from a nontranscribed region on chromosome VI. Middle, quantification of ChIP data, expressed as fold enrichment over background. (d) Recruitment of TAP-tagged wild-type Pcf11 and Pcf11 N107R to PMA1 was also monitored by ChIP assay; symbols as described in c.

  5. Cooperative binding of CID to phosphorylated CTD phosphoisoforms.
    Figure 5: Cooperative binding of CID to phosphorylated CTD phosphoisoforms.

    (a) Schematic of extended CTD phosphopeptides composed of four CTD heptad peptide repeats; phosphorylated serines (pSer), orange circles. (b) Chemical shift perturbations (CSP) of Rtt103 with the four-heptad repeat peptide phosphorylated at Ser2. Resonances showing increased CSPs with the four-repeat peptides, purple; resonances showing changes with both two- and four-repeat peptides, blue. (c) Residues showing chemical shift perturbations due to protein-protein interactions (purple) are primarily localized near the C terminus of helix 7; coloring as in b. (d,e) ChIP assays for the HA-tagged protein and Rpb3 from cells expressing wild-type HA-tagged Rtt103 (YSB2537) or Rtt103 E115R (YSB2538), in d; and wild-type HA-tagged Pcf11 (YSB2535) or Pcf11 D117A (YSB2536), in e. WT, wild type. Results are shown only for the poly(A) region, where Rtt103 or Pcf11 association is strongest (primer set 7 for PMA1 and 3 for ADH1). Error bars, s.d. from three repeats of the experiment.

  6. Cooperative model of Pcf11 and Rtt103 recruitment.
    Figure 6: Cooperative model of Pcf11 and Rtt103 recruitment.

    The density of Ser2P heptad repeats affects the location of transcription termination through cooperative recruitment of Pcf11 and Rtt103. At the poly(A) site, the density of Ser2P is highest15, allowing efficient recruitment of Pcf11 and Rtt103.

Accession codes

Primary accessions

Protein Data Bank


  1. Maniatis, T. & Reed, R. An extensive network of coupling among gene expression machines. Nature 416, 499506 (2002).
  2. Corden, J.L. & Patturajan, M.A. CTD function linking transcription to splicing. Trends Biochem. Sci. 22, 413416 (1997).
  3. Proudfoot, N.J., Furger, A. & Dye, M.J. Integrating mRNA processing with transcription. Cell 108, 501512 (2002).
  4. de la Mata, M. et al. A slow RNA polymerase II affects alternative splicing in vivo . Mol. Cell 12, 525532 (2003).
  5. Howe, K.J., Kane, C.M. & Ares, M. Jr. Perturbation of transcription elongation influences the fidelity of internal exon inclusion in Saccharomyces cerevisiae . RNA 9, 9931006 (2003).
  6. Ho, C.K. & Shuman, S. Distinct roles for CTD Ser-2 and Ser-5 phosphorylation in the recruitment and allosteric activation of mammalian mRNA capping enzyme. Mol. Cell 3, 405411 (1999).
  7. Bentley, D.L. Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr. Opin. Cell Biol. 17, 251256 (2005).
  8. Cho, E.J., Rodriguez, C.R., Takagi, T. & Buratowski, S. Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain. Genes Dev. 12, 34823487 (1998).
  9. Corden, J.L. Tails of RNA polymerase II. Trends Biochem. Sci. 15, 383387 (1990).
  10. Egloff, S. & Murphy, S. Cracking the RNA polymerase II CTD code. Trends Genet. 24, 280288 (2008).
  11. Phatnani, H.P. & Greenleaf, A.L. Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 20, 29222936 (2006).
  12. McCracken, S. et al. 5′-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. Genes Dev. 11, 33063318 (1997).
  13. Cho, E.J., Takagi, T., Moore, C.R. & Buratowski, S. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 11, 33193326 (1997).
  14. Komarnitsky, P., Cho, E.J. & Buratowski, S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14, 24522460 (2000).
  15. Ahn, S.H., Kim, M. & Buratowski, S. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3′ end processing. Mol. Cell 13, 6776 (2004).
  16. Chapman, R.D. et al. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 318, 17801782 (2007).
  17. Egloff, S. et al. Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression. Science 318, 17771779 (2007).
  18. Akhtar, M.S. et al. TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II. Mol. Cell 34, 387393 (2009).
  19. Stiller, J.W. & Cook, M.S. Functional unit of the RNA polymerase II C-terminal domain lies within heptapeptide pairs. Eukaryot. Cell 3, 735740 (2004).
  20. Vasiljeva, L., Kim, M., Mutschler, H., Buratowski, S. & Meinhart, A. The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain. Nat. Struct. Mol. Biol. 15, 795804 (2008).
  21. Kim, M. et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432, 517522 (2004).
  22. Becker, R., Loll, B. & Meinhart, A. 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, 2265922669 (2008).
  23. Sadowski, M., Dichtl, B., Hubner, W. & Keller, W. Independent functions of yeast Pcf11p in pre-mRNA 3′ end processing and in transcription termination. EMBO J. 22, 21672177 (2003).
  24. Meinhart, A. & Cramer, P. Recognition of RNA polymerase II carboxy-terminal domain by 3′-RNA-processing factors. Nature 430, 223226 (2004).
  25. Luo, W., Johnson, A.W. & Bentley, D.L. The role of Rat1 in coupling mRNA 3′-end processing to transcription termination: implications for a unified allosteric-torpedo model. Genes Dev. 20, 954965 (2006).
  26. Birse, C.E., Minvielle-Sebastia, L., Lee, B.A., Keller, W. & Proudfoot, N.J. Coupling termination of transcription to messenger RNA maturation in yeast. Science 280, 298301 (1998).
  27. Zhang, Z., Fu, J. & Gilmour, D.S. CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3′-end processing factor, Pcf11. Genes Dev. 19, 15721580 (2005).
  28. Kim, M., Ahn, S.H., Krogan, N.J., Greenblatt, J.F. & Buratowski, S. Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes. EMBO J. 23, 354364 (2004).
  29. Meinhart, A., Kamenski, T., Hoeppner, S., Baumli, S. & Cramer, P. A structural perspective of CTD function. Genes Dev. 19, 14011415 (2005).
  30. Buratowski, S. The CTD code. Nat. Struct. Biol. 10, 679680 (2003).
  31. Noble, C.G. et al. Key features of the interaction between Pcf11 CID and RNA polymerase II CTD. Nat. Struct. Mol. Biol. 12, 144151 (2005).
  32. Ramos, A. et al. The structure of the N-terminal domain of the fragile X mental retardation protein: a platform for protein-protein interaction. Structure 14, 2131 (2006).
  33. Noble, C.G., Walker, P.A., Calder, L.J. & Taylor, I.A. Rna14-Rna15 assembly mediates the RNA-binding capability of Saccharomyces cerevisiae cleavage factor IA. Nucleic Acids Res. 32, 33643375 (2004).
  34. Bai, Y. et al. Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors. Mol. Cell 25, 863875 (2007).
  35. Gudipati, R.K., Villa, T., Boulay, J. & Libri, D. Phosphorylation of the RNA polymerase II C-terminal domain dictates transcription termination choice. Nat. Struct. Mol. Biol. 15, 786794 (2008).
  36. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific LLC, San Carlos, California, USA, 2002).
  37. Sattler, M., Schleucher, J. & Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Mag. Res. Sp. 34, 93158 (1999).
  38. Zwahlen, C. et al. Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: Application to a bacteriophage lambda N-peptide/boxB RNA complex. J. Am. Chem. Soc. 119, 67116721 (1997).
  39. Farrow, N.A. et al. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 59846003 (1994).
  40. Guntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353378 (2004).
  41. Dominguez, C., Boelens, R. & Bonvin, A.M. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 17311737 (2003).
  42. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905921 (1998).
  43. Kim, M. et al. Distinct pathways for snoRNA and mRNA termination. Mol. Cell 24, 723734 (2006).

Download references

Author information


  1. Department of Biochemistry, University of Washington, Seattle, Washington, USA.

    • Bradley M Lunde &
    • Gabriele Varani
  2. Biomolecular Structure and Design Program, University of Washington, Seattle, Washington, USA.

    • Bradley M Lunde
  3. Department of Chemistry, University of Washington, Seattle, Washington, USA.

    • Steve L Reichow,
    • Thomas C Leeper,
    • Fan Yang &
    • Gabriele Varani
  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

    • Minkyu Kim,
    • Hyunsuk Suh &
    • Stephen Buratowski
  5. Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea.

    • Minkyu Kim
  6. Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg, Germany.

    • Hannes Mutschler &
    • Anton Meinhart


B.M.L. carried out all NMR titration and fluorescence anisotropy experiments with the four-heptad repeat CTD peptides. Diheptad repeat fluorescence anisotropy experiments were done by H.M. H.M. wrote the scripts for fitting the fluorescence anisotropy measurements. B.M.L. and H.M. analyzed the fluorescence anisotropy data. Structure determination of Rtt103-CID was done by S.L.R. Rtt103-CID bound to the Ser2P CTD was determined by B.M.L. Isotope-filtered NMR experiments were collected by T.C.L. All mutants were made by B.M.L., while F.Y. collected and analyzed NMR relaxation experiments. A.M. made the Pcf11-CID and Rtt103-CID constructs and developed the expression and purification conditions. S.B., M.K. and H.S. designed the in vivo ChIP experiments, and M.K. and H.S. constructed the strains and carried out the assays. B.M.L., S.L.R., H.S., S.B., A.M. and G.V. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (4M)

    Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods

Additional data