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:

NC2 mobilizes TBP on core promoter TATA boxes

Nature Structural & Molecular Biology volume 14, pages 1196–1201 (2007)Cite this article

Abstract

The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box–binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Förster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP–DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Loss of TBP–TATA protection against DNase I digestion mediated by NC2.
Figure 2: Half-life of TBP–NC2 on DNA.
Figure 3: The fluorescence intensity and FRET-efficiency traces of single TBP–DNA complexes, before, during and after NC2 addition.
Figure 4: Investigation of TBP–NC2 dynamics on DNA containing an H2B-J promoter.
Figure 5: CRIP analysis of TBP–TFIIA and TBP–NC2 complexes.
Figure 6: RCE analysis of TBP–NC2 complexes.
Figure 7: Model for TBP–NC2 complex dynamics and its consequences for preinitiation complex formation at TATA or altered preferred promoter recognition sites.

Similar content being viewed by others

References

  1. Stargell, L.A., Moqtaderi, Z., Dorris, D.R., Ogg, R.C. & Struhl, K. TFIIA has activator-dependent and core promoter functions in vivo. J. Biol. Chem. 275, 12374–12380 (2000).

    Article  CAS  Google Scholar 

  2. Buratowski, S., Hahn, S., Guarente, L. & Sharp, P.A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56, 549–561 (1989).

    Article  CAS  Google Scholar 

  3. Thomas, M.C. & Chiang, C.M. The general transcription machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 41, 105–178 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Albright, S.R. & Tjian, R. TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1–13 (2000).

    Article  CAS  Google Scholar 

  6. Lewis, B.A., Sims, R.J., III, Lane, W.S. & Reinberg, D. Functional characterization of core promoter elements: DPE-specific transcription requires the protein kinase CK2 and the PC4 coactivator. Mol. Cell 18, 471–481 (2005).

    Article  CAS  Google Scholar 

  7. Wright, K.J., Marr, M.T., II & Tjian, R. TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter. Proc. Natl. Acad. Sci. USA 103, 12347–12352 (2006).

    Article  CAS  Google Scholar 

  8. Sims, R.J., III, Mandal, S.S. & Reinberg, D. Recent highlights of RNA-polymerase-II-mediated transcription. Curr. Opin. Cell Biol. 16, 263–271 (2004).

    Article  CAS  Google Scholar 

  9. Meisterernst, M. & Roeder, R.G. Family of proteins that interact with TFIID and regulate promoter activity. Cell 67, 557–567 (1991).

    Article  CAS  Google Scholar 

  10. Inostroza, J.A., Mermelstein, F.H., Ha, I., Lane, W.S. & Reinberg, D. Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription. Cell 70, 477–489 (1992).

    Article  CAS  Google Scholar 

  11. Iratni, R. et al. Inhibition of excess nodal signaling during mouse gastrulation by the transcriptional corepressor DRAP1. Science 298, 1996–1999 (2002).

    Article  CAS  Google Scholar 

  12. Prelich, G. & Winston, F. Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo. Genetics 135, 665–676 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kamada, K. et al. Crystal structure of negative cofactor 2 recognizing the TBP-DNA transcription complex. Cell 106, 71–81 (2001).

    Article  CAS  Google Scholar 

  14. Goppelt, A., Stelzer, G., Lottspeich, F. & Meisterernst, M. A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains. EMBO J. 15, 3105–3116 (1996).

    Article  CAS  Google Scholar 

  15. Mermelstein, F. et al. Requirement of a corepressor for Dr1-mediated repression of transcription. Genes Dev. 10, 1033–1048 (1996).

    Article  CAS  Google Scholar 

  16. Gilfillan, S., Stelzer, G., Piaia, E., Hofmann, M.G. & Meisterernst, M. Efficient binding of NC2.TATA-binding protein to DNA in the absence of TATA. J. Biol. Chem. 280, 6222–6230 (2005).

    Article  CAS  Google Scholar 

  17. Albert, T.K. et al. Global distribution of negative cofactor 2 subunit-alpha on human promoters. Proc. Natl. Acad. Sci. USA 104, 10000–10005 (2007).

    Article  CAS  Google Scholar 

  18. Gadbois, E.L., Chao, D.M., Reese, J.C., Green, M.R. & Young, R.A. Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo. Proc. Natl. Acad. Sci. USA 94, 3145–3150 (1997).

    Article  CAS  Google Scholar 

  19. Xie, J., Collart, M., Lemaire, M., Stelzer, G. & Meisterernst, M. A single point mutation in TFIIA suppresses NC2 requirement in vivo. EMBO J. 19, 672–682 (2000).

    Article  CAS  Google Scholar 

  20. Chitikila, C., Huisinga, K.L., Irvin, J.D., Basehoar, A.D. & Pugh, B.F. Interplay of TBP inhibitors in global transcriptional control. Mol. Cell 10, 871–882 (2002).

    Article  CAS  Google Scholar 

  21. Geisberg, J.V., Holstege, F.C., Young, R.A. & Struhl, K. Yeast NC2 associates with the RNA polymerase II preinitiation complex and selectively affects transcription in vivo. Mol. Cell. Biol. 21, 2736–2742 (2001).

    Article  CAS  Google Scholar 

  22. Klejman, M.P. et al. NC2alpha interacts with BTAF1 and stimulates its ATP-dependent association with TATA-binding protein. Mol. Cell. Biol. 24, 10072–10082 (2004).

    Article  CAS  Google Scholar 

  23. Auty, R. et al. Purification of active TFIID from Saccharomyces cerevisiae. Extensive promoter contacts and co-activator function. J. Biol. Chem. 279, 49973–49981 (2004).

    Article  CAS  Google Scholar 

  24. Myong, S., Rasnik, I., Joo, C., Lohman, T.M. & Ha, T. Repetitive shuttling of a motor protein on DNA. Nature 437, 1321–1325 (2005).

    Article  CAS  Google Scholar 

  25. Amitani, I., Baskin, R.J. & Kowalczykowski, S.C. Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol. Cell 23, 143–148 (2006).

    Article  CAS  Google Scholar 

  26. Cox, J.M. et al. Bidirectional binding of the TATA box binding protein to the TATA box. Proc. Natl. Acad. Sci. USA 94, 13475–13480 (1997).

    Article  CAS  Google Scholar 

  27. Kapanidis, A.N. et al. Alternating-laser excitation of single molecules. Acc. Chem. Res. 38, 523–533 (2005).

    Article  CAS  Google Scholar 

  28. Kapanidis, A.N. et al. Retention of transcription initiation factor sigma70 in transcription elongation: single-molecule analysis. Mol. Cell 20, 347–356 (2005).

    Article  CAS  Google Scholar 

  29. Coban, O., Lamb, D.C., Zaychikov, E., Heumann, H. & Nienhaus, G.U. Conformational heterogeneity in RNA polymerase observed by single-pair FRET microscopy. Biophys. J. 90, 4605–4617 (2006).

    Article  CAS  Google Scholar 

  30. Kapanidis, A.N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).

    Article  Google Scholar 

  31. Coleman, R.A. & Pugh, B.F. Evidence for functional binding and stable sliding of the TATA binding protein on nonspecific DNA. J. Biol. Chem. 270, 13850–13859 (1995).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Goebel for excellent technical assistance, A. Lammens and D. Niessing for help with modeling of the TBP–DNA structure, T. Weil (Vanderbilt University) for providing the single-cysteine mutant of TBP, R.G. Roeder, L. Tora, P. Cramer and C. Bräuchle for crucial advice, and J. Michaelis (Ludwig Maximilian University Munich) for providing the MatLab software. This work was supported by grants from the Ludwig Maximilian University, Center for Nanoscience (CeNS), the German Excellence Initiative via the Nanosystems Initiative Munich and the Center for Integrated Protein Science (CiPS) Munich, Helmholtz Center Munich German Research Center for Environmental Health, and the Deutsche Forschungsgemeinschaft (SFB 646) to M.M. and D.C.L.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry and Center for Nanoscience (CeNS), Ludwig Maximilian University, Butenandtstr. 11, Munich, 81377, Germany

    Peter Schluesche & Don C Lamb

  2. Helmholtz Center Munich German Research Center for Environmental Health, Gene Expression Marchionini-Str. 25, 81377, Munich, Germany

    Gertraud Stelzer, Elisa Piaia & Michael Meisterernst

  3. Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, 61801, Illinois, USA

    Don C Lamb

  4. Department of Medicine, Institute for Cell and Tumor Biology, Westfalian Wilhelms University, Muenster, 48149, Germany

    Michael Meisterernst

Authors
  1. Peter Schluesche
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gertraud Stelzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elisa Piaia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Don C Lamb
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Meisterernst
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

P.S. built the single-molecule total internal reflection fluorescence microscope, performed all the single-molecule measurements, developed the analysis software and analyzed the single-molecule data. G.S. and E.P. performed biochemical analyses. D.C.L. and M.M. designed experiments and supervised the project.

Corresponding authors

Correspondence to Don C Lamb or Michael Meisterernst.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table, Supplementary Discussion, Supplementary Methods (PDF 891 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schluesche, P., Stelzer, G., Piaia, E. et al. NC2 mobilizes TBP on core promoter TATA boxes. Nat Struct Mol Biol 14, 1196–1201 (2007). https://doi.org/10.1038/nsmb1328

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1328

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