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.

Uniform transitions of the general RNA polymerase II transcription complex

Nature Structural & Molecular Biology volume 17, pages 1272–1278 (2010)Cite this article

Subjects

Abstract

We present genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms and transcription factors in proliferating yeast. Pol II exchanges initiation factors for elongation factors during a 5′ transition that is completed 150 nucleotides downstream of the transcription start site (TSS). The resulting elongation complex is composed of all the elongation factors and shows high levels of Ser7 and Ser5 phosphorylation on the C-terminal repeat domain (CTD) of Pol II. Ser2 phosphorylation levels increase until 600–1,000 nucleotides downstream of the TSS and do not correlate with recruitment of Spt6 and Pcf11, which bind the Ser2-phosphorylated CTD in vitro. This indicates CTD-independent recruitment mechanisms and CTD masking in vivo. Elongation complexes are productive and disassemble in a two-step 3′ transition. Paf1, Spt16 (part of the FACT complex), and the CTD kinases Bur1 and Ctk1 exit upstream of the polyadenylation site, whereas Spt4, Spt5, Spt6, Spn1 (also called Iws1) and Elf1 exit downstream. Transitions are uniform and independent of gene length, type and expression.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Genome-wide occupancy profiling of the Pol II machinery.
Figure 2: Statistical analysis indicates a general elongation complex.
Figure 3: Two-step 3′ transition observed at ribosomal protein (RP) genes.
Figure 4: Transcription complex composition and transitions are independent of gene length, expression and NFR size.
Figure 5: Pol II phosphorylation and factor occupancy.
Figure 6: Elongation complex occupancy predicts mRNA expression.

Accession codes

Accessions

ArrayExpress

References

  1. Pokholok, D.K., Hannett, N.M. & Young, R.A. Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9, 799–809 (2002).

    Google Scholar 

  2. Orphanides, G. & Reinberg, D. RNA polymerase II elongation through chromatin. Nature 407, 471–475 (2000).

    Google Scholar 

  3. Orphanides, G. & Reinberg, D. A unified theory of gene expression. Cell 108, 439–451 (2002).

    Google Scholar 

  4. 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 

  5. Schroeder, S.C., Schwer, B., Shuman, S. & Bentley, D. Dynamic association of capping enzymes with transcribing RNA polymerase II. Genes Dev. 14, 2435–2440 (2000).

    Google Scholar 

  6. Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol. Cell 36, 541–546 (2009).

    Google Scholar 

  7. Perales, R. & Bentley, D. “Cotranscriptionality”: the transcription elongation complex as a nexus for nuclear transactions. Mol. Cell 36, 178–191 (2009).

    Google Scholar 

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

    Google Scholar 

  9. Hirose, Y. & Manley, J.L. RNA polymerase II and the integration of nuclear events. Genes Dev. 14, 1415–1429 (2000).

    Google Scholar 

  10. Venters, B.J. & Pugh, B.F. A canonical promoter organization of the transcription machinery and its regulators in the Saccharomyces genome. Genome Res. 19, 360–371 (2009).

    Google Scholar 

  11. Aparicio, O. et al. Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. in Current Protocols in Molecular Biology (eds. Ausubel, F.A. et al.) Ch. 21, Unit 21.3 (Wiley, 2005).

  12. David, L. et al. A high-resolution map of transcription in the yeast genome. Proc. Natl. Acad. Sci. USA 103, 5320–5325 (2006).

    Google Scholar 

  13. Jasiak, A.J. et al. Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex. J. Biol. Chem. 283, 26423–26427 (2008).

    Google Scholar 

  14. Xu, Z. et al. Bidirectional promoters generate pervasive transcription in yeast. Nature 457, 1033–1037 (2009).

    Google Scholar 

  15. Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).

    Google Scholar 

  16. Dengl, S., Mayer, A., Sun, M. & Cramer, P. Structure and in vivo requirement of the yeast Spt6 SH2 domain. J. Mol. Biol. 389, 211–225 (2009).

    Google Scholar 

  17. Kuehner, J.N. & Brow, D.A. Quantitative analysis of in vivo initiator selection by yeast RNA polymerase II supports a scanning model. J. Biol. Chem. 281, 14119–14128 (2006).

    Google Scholar 

  18. Kostrewa, D. et al. RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature 462, 323–330 (2009).

    Google Scholar 

  19. Renner, D.B., Yamaguchi, Y., Wada, T., Handa, H. & Price, D.H. A highly purified RNA polymerase II elongation control system. J. Biol. Chem. 276, 42601–42609 (2001).

    Google Scholar 

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

    Google Scholar 

  21. Kim, M., Suh, H., Cho, E.-J. & Buratowski, S. Phosphorylation of the yeast Rpb1 C-terminal domain at serines 2, 5, and 7. J. Biol. Chem. 284, 26421–26426 (2009).

    Google Scholar 

  22. 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 

  23. Boeing, S., Rigault, C., Heidemann, M., Eick, D. & Meisterernst, M. RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a Mediator-dependent fashion. J. Biol. Chem. 285, 188–196 (2010).

    Google Scholar 

  24. Murray, S., Udupa, R., Yao, S., Hartzog, G. & Prelich, G. Phosphorylation of the RNA polymerase II carboxy-terminal domain by the Bur1 cyclin-dependent kinase. Mol. Cell. Biol. 21, 4089–4096 (2001).

    Google Scholar 

  25. Liu, Y. et al. Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of the PAF complex. Mol. Cell. Biol. 29, 4852–4863 (2009).

    Google Scholar 

  26. Rodriguez, C.R. et al. Kin28, the TFIIH-Associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II. Mol. Cell. Biol. 20, 104–112 (2000).

    Google Scholar 

  27. 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. 21, 160–174 (2007).

    Google Scholar 

  28. Barillà, D., Lee, B.A. & Proudfoot, N.J. Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 98, 445–450 (2001).

    Google Scholar 

  29. Nechaev, S. et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335–338 (2010).

    Google Scholar 

  30. Zeitlinger, J. et al. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat. Genet. 39, 1512–1516 (2007).

    Google Scholar 

  31. Core, L.J., Waterfall, J.J. & Lis, J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

    Google Scholar 

  32. Rahl, P.B. et al. c-Myc regulates transcriptional pause release. Cell 141, 432–445 (2010).

    Google Scholar 

  33. Jiang, C. & Pugh, B.F. Nucleosome positioning and gene regulation: advances through genomics. Nat. Rev. Genet. 10, 161–172 (2009).

    Google Scholar 

  34. Radonjic, M. et al. Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit. Mol. Cell 18, 171–183 (2005).

    Google Scholar 

  35. Chen, H.-T., Warfield, L. & Hahn, S. The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex. Nat. Struct. Mol. Biol. 14, 696–703 (2007).

    Google Scholar 

  36. Hirtreiter, A. et al. Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif. Nucleic Acids Res. 38, 4040–4051 (2010).

    Google Scholar 

  37. Guo, M. et al. Core structure of the yeast Spt4-Spt5 complex: a conserved module for regulation of transcription elongation. Structure 16, 1649–1658 (2008).

    Google Scholar 

  38. Lindstrom, D.L. et al. Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell. Biol. 23, 1368–1378 (2003).

    Google Scholar 

  39. Krogan, N.J. et al. RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22, 6979–6992 (2002).

    Google Scholar 

  40. Prather, D., Krogan, N.J., Emili, A., Greenblatt, J.F. & Winston, F. Identification and characterization of Elf1, a conserved transcription elongation factor in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 10122–10135 (2005).

    Google Scholar 

  41. Qiu, H., Hu, C. & Hinnebusch, A.G. Phosphorylation of the Pol II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters. Mol. Cell 33, 752–762 (2009).

    Google Scholar 

  42. Zhou, K., Kuo, W.H.W., Fillingham, J. & Greenblatt, J.F. Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5. Proc. Natl. Acad. Sci. USA 106, 6956–6961 (2009).

    Google Scholar 

  43. Laribee, R.N. et al. BUR kinase selectively regulates H3 K4 trimethylation and H2B ubiquitylation through recruitment of the PAF elongation complex. Curr. Biol. 15, 1487–1493 (2005).

    Google Scholar 

  44. Stuwe, T. et al. The FACT Spt16 “peptidase” domain is a histone H3–H4 binding module. Proc. Natl. Acad. Sci. USA 105, 8884–8889 (2008).

    Google Scholar 

  45. Belotserkovskaya, R. et al. FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093 (2003).

    Google Scholar 

  46. Ahn, S.H., Keogh, M.-C. & Buratowski, S. Ctk1 promotes dissociation of basal transcription factors from elongating RNA polymerase II. EMBO J. 28, 205–212 (2009).

    Google Scholar 

  47. 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 

  48. Keogh, M.-C., Podolny, V. & Buratowski, S. Bur1 kinase is required for efficient transcription elongation by RNA polymerase II. Mol. Cell. Biol. 23, 7005–7018 (2003).

    Google Scholar 

  49. Glover-Cutter, K., Kim, S., Espinosa, J. & Bentley, D.L. RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes. Nat. Struct. Mol. Biol. 15, 71–78 (2008).

    Google Scholar 

  50. 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. 280, 913–922 (2005).

    Google Scholar 

  51. Kaplan, C.D., Laprade, L. & Winston, F. Transcription elongation factors repress transcription initiation from cryptic sites. Science 301, 1096–1099 (2003).

    Google Scholar 

  52. 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 

  53. Hesselberth, J.R. et al. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat. Methods 6, 283–289 (2009).

    Google Scholar 

  54. Yuan, G.C. et al. Genome-scale identification of nucleosome positions in S. cerevisiae. Science 309, 626–630 (2005).

    Google Scholar 

  55. Fan, X., Lamarre-Vincent, N., Wang, Q. & Struhl, K. Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments. Nucleic Acids Res. 36, e125 (2008).

    Google Scholar 

Download references

Acknowledgements

We thank H. Feldmann and D. Martin for help, D. Eick (Helmholtz Zentrum München) for providing antibodies and A. Tresch for discussions. J.S. was supported by the Deutsche Forschungsgemeinschaft and SFB646. P.C. was supported by the Deutsche Forschungsgemeinschaft, the SFB646, the TR5, the Nanosystems Initiative Munich NIM, the Elitenetzwerk Bayern, the Jung-Stiftung and the Fonds der Chemischen Industrie.

Author information

Author notes

  1. Andreas Mayer, Michael Lidschreiber and Matthias Siebert: These authors contributed equally to this work.

Authors and Affiliations

  1. Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science, Ludwig-Maximilians-Universität München, Munich, Germany

    Andreas Mayer, Michael Lidschreiber, Matthias Siebert, Kristin Leike, Johannes Söding & Patrick Cramer

Authors
  1. Andreas Mayer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Lidschreiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias Siebert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kristin Leike
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johannes Söding
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrick Cramer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.M. established experimental protocols; A.M., M.L. and K.L. performed experiments; M.L. and M.S. evaluated data sets; J.S. designed and supervised data evaluation; P.C. designed and supervised research and wrote the manuscript.

Corresponding authors

Correspondence to Johannes Söding or Patrick Cramer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 5384 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mayer, A., Lidschreiber, M., Siebert, M. et al. Uniform transitions of the general RNA polymerase II transcription complex. Nat Struct Mol Biol 17, 1272–1278 (2010). https://doi.org/10.1038/nsmb.1903

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1903

This article is cited by

Search

Advanced 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