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.

Paused RNA polymerase II inhibits new transcriptional initiation

Abstract

RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. However, it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: ChIP-nexus detects the precise locations of basal transcription factors and Pol II along the core promoter in vivo.
Figure 2: Measurement of the half-lives of paused Pol II across the genome using triptolide treatment.
Figure 3: Promoters with stable Pol II pausing lack PICs but show downstream occupancy of TBP.
Figure 4: Paused Pol II inhibits new initiation.

Accession codes

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

Protein Data Bank

Sequence Read Archive

References

  1. 1

    Sainsbury, S., Bernecky, C. & Cramer, P. Structural basis of transcription initiation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 129–143 (2015).

    CAS  PubMed  Google Scholar 

  2. 2

    Darzacq, X. et al. Imaging transcription in living cells. Annu. Rev. Biophys. 38, 173–196 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Nogales, E., Louder, R.K. & He, Y. Cryo-EM in the study of challenging systems: the human transcription pre-initiation complex. Curr. Opin. Struct. Biol. 40, 120–127 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Conaway, R.C. & Conaway, J.W. General initiation factors for RNA polymerase II. Annu. Rev. Biochem. 62, 161–190 (1993).

    CAS  PubMed  Google Scholar 

  5. 5

    Roeder, R.G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 (1996).

    CAS  Google Scholar 

  6. 6

    Tirode, F., Busso, D., Coin, F. & Egly, J.M. Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7. Mol. Cell 3, 87–95 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Holstege, F.C., van der Vliet, P.C. & Timmers, H.T. Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH. EMBO J. 15, 1666–1677 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Adelman, K. & Lis, J.T. Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans. Nat. Rev. Genet. 13, 720–731 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Jonkers, I. & Lis, J.T. Getting up to speed with transcription elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 167–177 (2015).

    CAS  Article  Google Scholar 

  10. 10

    Hendrix, D.A., Hong, J.W., Zeitlinger, J., Rokhsar, D.S. & Levine, M.S. Promoter elements associated with RNA Pol II stalling in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 105, 7762–7767 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    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).

    CAS  Google Scholar 

  12. 12

    Lagha, M. et al. Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo. Cell 153, 976–987 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Boettiger, A.N. & Levine, M. Synchronous and stochastic patterns of gene activation in the Drosophila embryo. Science 325, 471–473 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Gilchrist, D.A. et al. Pausing of RNA polymerase II disrupts DNA-specified nucleosome organization to enable precise gene regulation. Cell 143, 540–551 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Li, J. et al. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing. Mol. Cell 50, 711–722 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Kwak, H., Fuda, N.J., Core, L.J. & Lis, J.T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Rhee, H.S. & Pugh, B.F. Comprehensive genome-wide protein–DNA interactions detected at single-nucleotide resolution. Cell 147, 1408–1419 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Pugh, B.F. & Venters, B.J. Genomic organization of human transcription initiation complexes. PLoS One 11, e0149339 (2016).

    PubMed  PubMed Central  Google Scholar 

  21. 21

    He, Q., Johnston, J. & Zeitlinger, J. ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat. Biotechnol. 33, 395–401 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Titov, D.V. et al. XPB, a subunit of TFIIH, is a target of the natural product triptolide. Nat. Chem. Biol. 7, 182–188 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Geiger, J.H., Hahn, S., Lee, S. & Sigler, P.B. Crystal structure of the yeast TFIIA/TBP/DNA complex. Science 272, 830–836 (1996).

    CAS  PubMed  Google Scholar 

  24. 24

    Tan, S., Hunziker, Y., Sargent, D.F. & Richmond, T.J. Crystal structure of a yeast TFIIA/TBP/DNA complex. Nature 381, 127–151 (1996).

    CAS  PubMed  Google Scholar 

  25. 25

    Imbalzano, A.N., Zaret, K.S. & Kingston, R.E. Transcription factor (TF) IIB and TFIIA can independently increase the affinity of the TATA-binding protein for DNA. J. Biol. Chem. 269, 8280–8286 (1994).

    CAS  PubMed  Google Scholar 

  26. 26

    He, Y. et al. Near-atomic resolution visualization of human transcription promoter opening. Nature 533, 359–365 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Horn, A.E., Kugel, J.F. & Goodrich, J.A. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity. Nucleic Acids Res. 44, 7132–7143 (2016).

    PubMed  PubMed Central  Google Scholar 

  28. 28

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

    CAS  Google Scholar 

  29. 29

    Rhee, H.S. & Pugh, B.F. Genome-wide structure and organization of eukaryotic pre-initiation complexes. Nature 483, 295–301 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Plaschka, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016).

    CAS  PubMed  Google Scholar 

  31. 31

    Burley, S.K. & Roeder, R.G. Biochemistry and structural biology of transcription factor IID (TFIID). Annu. Rev. Biochem. 65, 769–799 (1996).

    CAS  PubMed  Google Scholar 

  32. 32

    Louder, R.K. et al. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly. Nature 531, 604–609 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Verrijzer, C.P., Chen, J.L., Yokomori, K. & Tjian, R. Binding of TAFs to core elements directs promoter selectivity by RNA polymerase II. Cell 81, 1115–1125 (1995).

    CAS  Article  Google Scholar 

  34. 34

    Jonkers, I., Kwak, H. & Lis, J.T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. eLife 3, e02407 (2014).

    PubMed  PubMed Central  Google Scholar 

  35. 35

    Chen, F., Gao, X. & Shilatifard, A. Stably paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor triptolide. Genes Dev. 29, 39–47 (2015).

    PubMed  PubMed Central  Google Scholar 

  36. 36

    Grünberg, S., Warfield, L. & Hahn, S. Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening. Nat. Struct. Mol. Biol. 19, 788–796 (2012).

    PubMed  PubMed Central  Google Scholar 

  37. 37

    Alekseev, S. et al. Transcription without XPB establishes a unified helicase-independent mechanism of promoter opening in eukaryotic gene expression. Mol. Cell 65, 504–514 (2017).

    CAS  PubMed  Google Scholar 

  38. 38

    Zhang, Z. et al. Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis. Genes Dev. 30, 2106–2118 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Henriques, T. et al. Stable pausing by RNA polymerase II provides an opportunity to target and integrate regulatory signals. Mol. Cell 52, 517–528 (2013).

    CAS  PubMed  Google Scholar 

  41. 41

    Buckley, M.S., Kwak, H., Zipfel, W.R. & Lis, J.T. Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation. Genes Dev. 28, 14–19 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Chen, K. et al. A global change in RNA polymerase II pausing during the Drosophila midblastula transition. eLife 2, e00861 (2013).

    PubMed  PubMed Central  Google Scholar 

  43. 43

    Gaertner, B. et al. Poised RNA polymerase II changes over developmental time and prepares genes for future expression. Cell Reports 2, 1670–1683 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Zawel, L., Kumar, K.P. & Reinberg, D. Recycling of the general transcription factors during RNA polymerase II transcription. Genes Dev. 9, 1479–1490 (1995).

    CAS  PubMed  Google Scholar 

  45. 45

    Yudkovsky, N., Ranish, J.A. & Hahn, S. A transcription reinitiation intermediate that is stabilized by activator. Nature 408, 225–229 (2000).

    CAS  PubMed  Google Scholar 

  46. 46

    Van Dyke, M.W., Sawadogo, M. & Roeder, R.G. Stability of transcription complexes on class II genes. Mol. Cell. Biol. 9, 342–344 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Chao, S.H. & Price, D.H. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J. Biol. Chem. 276, 31793–31799 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Marshall, N.F. & Price, D.H. Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J. Biol. Chem. 270, 12335–12338 (1995).

    CAS  PubMed  Google Scholar 

  49. 49

    Saeki, H. & Svejstrup, J.Q. Stability, flexibility, and dynamic interactions of colliding RNA polymerase II elongation complexes. Mol. Cell 35, 191–205 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Ehrensberger, A.H., Kelly, G.P. & Svejstrup, J.Q. Mechanistic interpretation of promoter-proximal peaks and RNAPII density maps. Cell 154, 713–715 (2013).

    CAS  PubMed  Google Scholar 

  51. 51

    Bothma, J.P. et al. Dynamic regulation of eve stripe 2 expression reveals transcriptional bursts in living Drosophila embryos. Proc. Natl. Acad. Sci. USA 111, 10598–10603 (2014).

    CAS  PubMed  Google Scholar 

  52. 52

    Fukaya, T., Lim, B. & Levine, M. Enhancer control of transcriptional bursting. Cell 166, 358–368 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Suter, D.M. et al. Mammalian genes are transcribed with widely different bursting kinetics. Science 332, 472–474 (2011).

    CAS  Google Scholar 

  54. 54

    Zoller, B., Nicolas, D., Molina, N. & Naef, F. Structure of silent transcription intervals and noise characteristics of mammalian genes. Mol. Syst. Biol. 11, 823 (2015).

    PubMed  PubMed Central  Google Scholar 

  55. 55

    Gilchrist, D.A. et al. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly. Genes Dev. 22, 1921–1933 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Deal, R.B., Henikoff, J.G. & Henikoff, S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science 328, 1161–1164 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Pedraza, J.M. & Paulsson, J. Effects of molecular memory and bursting on fluctuations in gene expression. Science 319, 339–343 (2008).

    CAS  PubMed  Google Scholar 

  58. 58

    Coulon, A., Chow, C.C., Singer, R.H. & Larson, D.R. Eukaryotic transcriptional dynamics: from single molecules to cell populations. Nat. Rev. Genet. 14, 572–584 (2013).

    CAS  PubMed  Google Scholar 

  59. 59

    Ghavi-Helm, Y. et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512, 96–100 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank P. Verrijzer (Erasmus University Medical Center) for TAF2 antibodies, J. Kadonaga (University of California, San Diego) for TFIIA, TFIIB, TBP and TFIIF antibodies, J. Johnston for help with data analysis, and R. Krumlauf, R. Conaway, J. Conaway, C. Kaplan and R. Fropf for comments on the manuscript. The work was funded by the Stowers Institute for Medical Research.

Author information

Affiliations

Authors

Contributions

W.S. and J.Z. conceived the project, W.S. performed all experiments and computational analyses, and W.S. and J.Z. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Julia Zeitlinger.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 2–4 (PDF 9955 kb)

Supplementary Table 1

Summary of paused Pol II half-lives across the genome. (XLS 718 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shao, W., Zeitlinger, J. Paused RNA polymerase II inhibits new transcriptional initiation. Nat Genet 49, 1045–1051 (2017). https://doi.org/10.1038/ng.3867

Download citation

Further reading

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