Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Sequence alignment of CIDs and diheptad CTD phosphopeptides used in this study.
Figure 2: Binding of Rtt103-CID and Pcf11-CID to diheptad CTD phosphopeptides monitored by fluorescence anisotropy and NMR.
Figure 3: Structure of Rtt103-CID and recognition of the Ser2P CTD.
Figure 4: CTD specificity can be altered by a single-residue change.
Figure 5: Cooperative binding of CID to phosphorylated CTD phosphoisoforms.
Figure 6: Cooperative model of Pcf11 and Rtt103 recruitment.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Maniatis, T. & Reed, R. An extensive network of coupling among gene expression machines. Nature 416, 499–506 (2002).

    Google Scholar 

  2. Corden, J.L. & Patturajan, M.A. CTD function linking transcription to splicing. Trends Biochem. Sci. 22, 413–416 (1997).

    Google Scholar 

  3. Proudfoot, N.J., Furger, A. & Dye, M.J. Integrating mRNA processing with transcription. Cell 108, 501–512 (2002).

    Google Scholar 

  4. de la Mata, M. et al. A slow RNA polymerase II affects alternative splicing in vivo. Mol. Cell 12, 525–532 (2003).

    Google Scholar 

  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, 993–1006 (2003).

    Google Scholar 

  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, 405–411 (1999).

    Google Scholar 

  7. Bentley, D.L. Rules of engagement: co-transcriptional recruitment of pre-mRNA processing factors. Curr. Opin. Cell Biol. 17, 251–256 (2005).

    Google Scholar 

  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, 3482–3487 (1998).

    Google Scholar 

  9. Corden, J.L. Tails of RNA polymerase II. Trends Biochem. Sci. 15, 383–387 (1990).

    Google Scholar 

  10. Egloff, S. & Murphy, S. Cracking the RNA polymerase II CTD code. Trends Genet. 24, 280–288 (2008).

    Google Scholar 

  11. Phatnani, H.P. & Greenleaf, A.L. Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 20, 2922–2936 (2006).

    Google Scholar 

  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, 3306–3318 (1997).

    Google Scholar 

  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, 3319–3326 (1997).

    Google Scholar 

  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, 2452–2460 (2000).

    Google Scholar 

  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, 67–76 (2004).

    Google Scholar 

  16. Chapman, R.D. et al. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 318, 1780–1782 (2007).

    Google Scholar 

  17. Egloff, S. et al. Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression. Science 318, 1777–1779 (2007).

    Google Scholar 

  18. Akhtar, M.S. et al. TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II. Mol. Cell 34, 387–393 (2009).

    Google Scholar 

  19. Stiller, J.W. & Cook, M.S. Functional unit of the RNA polymerase II C-terminal domain lies within heptapeptide pairs. Eukaryot. Cell 3, 735–740 (2004).

    Google Scholar 

  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, 795–804 (2008).

    Google Scholar 

  21. Kim, M. et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432, 517–522 (2004).

    Google Scholar 

  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, 22659–22669 (2008).

    Google Scholar 

  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, 2167–2177 (2003).

    Google Scholar 

  24. Meinhart, A. & Cramer, P. Recognition of RNA polymerase II carboxy-terminal domain by 3′-RNA-processing factors. Nature 430, 223–226 (2004).

    Google Scholar 

  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, 954–965 (2006).

    Google Scholar 

  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, 298–301 (1998).

    Google Scholar 

  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, 1572–1580 (2005).

    Google Scholar 

  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, 354–364 (2004).

    Google Scholar 

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

    Google Scholar 

  30. Buratowski, S. The CTD code. Nat. Struct. Biol. 10, 679–680 (2003).

    Google Scholar 

  31. Noble, C.G. et al. Key features of the interaction between Pcf11 CID and RNA polymerase II CTD. Nat. Struct. Mol. Biol. 12, 144–151 (2005).

    Google Scholar 

  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, 21–31 (2006).

    Google Scholar 

  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, 3364–3375 (2004).

    Google Scholar 

  34. Bai, Y. et al. Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors. Mol. Cell 25, 863–875 (2007).

    Google Scholar 

  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, 786–794 (2008).

    Google Scholar 

  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, 93–158 (1999).

    Google Scholar 

  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, 6711–6721 (1997).

    Google Scholar 

  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, 5984–6003 (1994).

    Google Scholar 

  40. Guntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    Google Scholar 

  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, 1731–1737 (2003).

    Google Scholar 

  42. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Google Scholar 

  43. Kim, M. et al. Distinct pathways for snoRNA and mRNA termination. Mol. Cell 24, 723–734 (2006).

    Google Scholar 

Download references

Acknowledgements

We thank R. Becker for his assistance with the fluorescence anisotropy experiments. We also thank B. Demeler and V. Schirf at the Center for Analytical Ultracentrifugation of Macromolecular Assemblies for assistance with the AUC experiments. This work was supported by grants from the National Institute of General Medical Sciences of the US National Institutes of Health to G.V. (GM64440) and S.B. (GM56663) and from the German Research Foundation to A.M. (ME3135/1-1) as well as by the WCU project (305-20080089) from KMEST and grants from the National Research Foundation of Korea (NRF) (R31-2009-000-10032-0 and 2010-0011750) to M.K. A portion of the research was performed using the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research located at Pacific Northwest National Laboratory.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Gabriele Varani.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods (PDF 3888 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lunde, B., Reichow, S., Kim, M. et al. Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nat Struct Mol Biol 17, 1195–1201 (2010). https://doi.org/10.1038/nsmb.1893

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1893

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing