Skip to main content
SpringerLink
Log in

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

Abstract

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10−15–10−10 m) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, γ, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pm. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bezkurov, S.M., Vodyanoy, L., Parsegian, V.A. 1994. Counting polymers moving through a single ion channel. Nature 370:279–281

    Google Scholar 

  • Binning, G., Quate, C.F., Gerber, Ch. 1986. Atomic force microscope. Phys. Rev. Lett. 56:930–933

    Google Scholar 

  • Boulikas, T. 1994. Putative nuclear localization signals (NLS) in protein transcription factors. J. Cell. Biochem. 55:32–58

    Google Scholar 

  • Braunstein, D., Spudich, A. 1994. Structure and activation dynamics of RBL-2H3 cells observed with scanning force microscopy. Biophys. J. 66:1717–172

    Google Scholar 

  • Buratowski, S. 1994. The basics of basal transcription of RNA polymerase II. Cell 77:1–3

    Google Scholar 

  • Bustamante, J.O. 1992. Nuclear ion channels in cardiac myocytes. Pfluegers Arch. 421:473–485

    Google Scholar 

  • Bustamante, J.O. 1993. Restricted ion flow at the nuclear envelope of cardiac myocytes. Biophys. J. 64:1735–1749

    Google Scholar 

  • Bustamante, J.O. 1994a. Open states of nuclear envelope ion channels in cardiac myocytes. J. Membrane Biol. 138:77–89

    Google Scholar 

  • Bustamante, J.O. 1994b. Nuclear electrophysiology. J. Membrane Biol. 138:105–112

    Google Scholar 

  • Bustamante, J.O., Hanover, J.A., Liepins, A. 1995a. The ion channel behavior of the nuclear pore complex. J. Membrane Biol. 146:

  • Bustamante, J.O., Liepins, A., Hanover, J.A. 1994. Nuclear pore complex ion channels. Mol. Membr. Biol. 11:141–150

    Google Scholar 

  • Bustamante, J.O., Oberleithner, H., Hanover, J.A., Liepins, A. 1995b. Patch-clamp detection of transcription factor translocation along the nuclear pore complex channel. J. Membrane Biol. 146:

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

    Google Scholar 

  • Davis, L.I., Blobel, G. 1987. Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway. Proc. Natl. Acad. Sci. USA 84:7552–7556

    Google Scholar 

  • Engel, A. 1991. Biological applications of scanning probe microscopes. Annu. Rev. Biophys. Chem. 20:79–108

    Google Scholar 

  • Greenblatt, J. 1992. Riding high on the TATA box. Nature 360:16–17

    Google Scholar 

  • Haltiwanger, R.S., Blomberg, M., Hart, G.W. 1992. Glycosylation of nuclear and cytoplasmic proteins. J. Biol. Chem. 267:9005–9013

    Google Scholar 

  • Haltiwanger, R.S., Kelly, W.G., Roquemore, E.P., Blomberg, M., Dong, L.-Y.D., Kreppel, L., Chou, T.-Y., Hart, G.W. 1992. Glycosylation of nuclear and cytoplasmic proteins is ubiquitous and dynamic. Biochem. Soc. Trans. 20:264–269

    Google Scholar 

  • Hart, G.W., Haltiwanger, R.S., Holt, G.D., Kelly, W.G. 1989. Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 58:841–874

    Google Scholar 

  • Hernandez, N. 1993. TBP, a universal eukaryotic transcription factor? Genes Dev. 7:1291–1308

    Google Scholar 

  • Hofman, M. 1993. The cell's nucleus shapes up. Science 259:1257–1259

    Google Scholar 

  • Hoh, J.H., Hansma, P.K. 1992. Atomic force microscopy for high-resolution imaging in cell biology. Trends Cell Biol. 2:208–213

    Google Scholar 

  • Hoh, J.H., Lal, R., John, S.A., Revel, J.-P., Arnsdorf, M.F. 1991. Atomic force microscopy and dissection of gap junctions. Science 253:1405–1408

    Google Scholar 

  • Hoh, J.H., Sosinsky, G.E., Revel, J.-P., Hansma, P.K. 1993. Structure of the extracellular surface of the gap junction by atomic force microscopy. Biophys. J. 65:149–163

    Google Scholar 

  • Holt, G.D., Snow, C.M., Senior, A., Haltiwanger, R.S., Gerace, L., Hart, G.W. 1987. Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell. Biol. 104:1157–1164

    Google Scholar 

  • Hori, R., Carey, M. 1994. The role of activators in assembly of RNA polymerase II transcription complexes. Curr. Opin. Gen. Dev. 4:236–244

    Google Scholar 

  • Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, P.A., Roeder, R.G. 1989. Cloning and structure of a yeast gene encoding a general transcription initiation factor TFIID that binds to the TATA box. Nature 341:299–303

    Google Scholar 

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

    Google Scholar 

  • Jackson, S.P., Tjian, R. 1988. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell 55:125–133

    Google Scholar 

  • Kato, K., Makino, Y., Kishimoto, T., Yamauchi, J., Kato, S., Muramatsu, M., Tamura, T. 1994. Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain. Nucleic Acids Res. 22:1179–1185

    Google Scholar 

  • Kim, J.L., Burley, S.K. 1994. 1.8 A resolution refined structure of TBP recognizing the minor groove of TATAAAAG. Struct. Biol. 1:638–653

    Google Scholar 

  • Klein, C., Strahl, K., 1994. Increased recruitment of TATA-binding protein to the promoter by transcriptional activation domains in vivo. Science 266:280–282

    Google Scholar 

  • Kokubo, T., Gong, D.W., Wooton, J.C., Hoikoshi, M., Roeder, R.G., Nakatani, Y. 1994. Molecular cloning of Drosophila TFIID subunits. Nature 364:484–487

    Google Scholar 

  • Lal, R., John, S.A. 1994. Biological applications of atomic force microscopy. Am. J. Physiol. 266:C1-C21

    Google Scholar 

  • Lal, R., Kim, H., Garavito, R.M., Arnsdorf, M.F. 1993. Imaging of reconstituted biological channels at molecular resolution by atomic force microscopy. Am. J. Physiol. 265:C851-C856

    Google Scholar 

  • Lal, R., Yu, L. 1993. Atomic force microscopy of cloned nicotinic acetylcholine receptor expressed in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 90:7280–7284

    Google Scholar 

  • Lewin, B. 1990. Commitment and activation at pol II promoters. Cell 61:1161–1164

    Google Scholar 

  • Lian, J.B., Stein, G.S., Bortell, R., Owen, T.A. 1991. Phenotype suppression: a postulated molecular mechanism for mediating the relationship of proliferation and differentiation by Fos/Jun interactions at AP-1 sites in steroid responsive promoter elements of tissue-specific genes. J. Cell Biochem. 45:9–14

    Google Scholar 

  • Manivannan, K., Ramanan, S.V., Mathias, R.T., Brink, P.R. 1992. Multichannel recordings from membranes which contain gap junctions. Biophys J. 61:216–227

    Google Scholar 

  • Marx, J. 1993. Forging a path to the nucleus. Science 260:1588–1560

    Google Scholar 

  • Miller, M., Park, M.K., Hanover, J.A. 1991. Nuclear pore complex: structure, function and regulation. Physiol. Rev. 71:681–686

    Google Scholar 

  • Morris, V.J. 1994. Biological applications of scanning probe microscopies. Prog. Biophys. Mol. Biol. 61:131–185

    Google Scholar 

  • Nebert, D.W. 1994. Drug-metabolizing enzymes in ligand modulated transcription. Biochem. Pharmacol. 47:25–37

    Google Scholar 

  • Nikolov, D.B., Burley, S.K. 1994. 2.1 A resolution refined structure of a TATA box-binding protein (TBP). Struct. Biol. 1:621–637

    Google Scholar 

  • Oberleithner, H., Birnckmann, H., Schwab, A., Khrone, G. 1994. Imaging nuclear pores of aldosterone sensitive kidney cells by atomic force microscopy. Proc. Natl. Acad. Sci. USA 91:9784–9788

    Google Scholar 

  • Oberleithner, H., Giebisch, G., Geibel, J. 1993. Imaging the lamellipodium of migrating epithelial cells in vivo by atomic force microscopy. Pfluegers Arch. 425:401–407

    Google Scholar 

  • Panté, N., Aebi, U. 1994. Towards understanding the three-dimensional structure of the nuclear pore complex at the molecular level. Curr. Opin. Struct. Biol 4:187–196

    Google Scholar 

  • Parker, T.G., Schneider, M.D. 1991. Growth factors, proto-oncogenes, and plasticity of the cardiac phenotype. Annu. Rev. Physiol. 53:179–200

    Google Scholar 

  • Peterson, M.G., Tanese, N., Pugh, B.F., Tjian, R. 1990. Functional domains and upstream activation properties of cloned human TATA binding protein. Science 248:1625–1630

    Google Scholar 

  • Peterson, M.G., Tupy, J.L. 1994. Transcription factors: a new frontier in pharmaceutical development. Biochem. Pharmacol. 47:127–128

    Google Scholar 

  • Prabhakar, P., Kayastha, A.M. 1994. Mechanism of DNA-drug interactions. Appl. Biochem. Biotech. 47:3955

    Google Scholar 

  • Radmacher, M., Tillmann, R.W., Firtz, N., Gaub, H.E. 1992. From molecules to cells-imaging soft samples with the AFM. Science 257:1900–1905

    Google Scholar 

  • Ramanan, S.V., Brink, P.R. Multichannel recordings from membranes which contain gap junctions. II. Substates and conductance shifts. Biophys. J. 65:1387–1395

  • Rutgar, D., Hansma, P.K. 1990. Atomic force microscopy. Phys. Today 43:23–30

    Google Scholar 

  • Rowlands, T., Baumann, P., Jackson, S.P. 1994. The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Science 264:1326–1329

    Google Scholar 

  • Sadoshima, J.-I., Jahn, L., Takahashi, T., Kulik, T.J., Izumo, S. 1992. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells: an in vitro model of load-induced cardiac hypertrophy. J. Biol Chem. 267:10551–10560

    Google Scholar 

  • Simon, S.M., Blobel, G. 1991. A protein-conducting channel in the endoplasmic reticulum. Cell 65:371–380

    Google Scholar 

  • Simon, S.M., Blobel, G. 1992. Signal peptides open protein-conducting channels in E. coli. Cell 69:677–684

    Google Scholar 

  • Strahl, K. 1994. Duality of TBP, the universal transcription factor. Science 263:1103–1104

    Google Scholar 

  • Sweillens, S., Pirson, I. 1994. Highly sensitive control of transcriptional actiaction by factor heterodimerization. Biochem. J. 301:9–12

    Google Scholar 

  • Tanese, N., Tjian, R. 1993. Coactivators and TAFs: a new class of eukaryotic transcription factors that connect activators to the basal machinery. Cold Spring Harbor Symp. Quant. Biol. 43:179–185

    Google Scholar 

  • Tjian, R., Maniatis, T. 1994. Transcriptional activation: a complex puzzle with few easy pieces. Cell 77:5–8

    Google Scholar 

  • Weis, L., Reinberg, D. 1992. Transcription by RNA polymerase II: initiator-directed formation of transcription-competent complexes. FASEB J 6:3300–3309

    Google Scholar 

  • Wolffe, A.P. 1994. Architectural transcription factors. Science 264:1100–1101

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Physiology, University of Maryland School of Medicine, 21201, Baltimore, MD

    J. O. Bustamante

  2. Division of Basic Medical Sciences, Memorial University of Newfoundland, School of Medicine, A1B 3V6, St. John's, Newfoundland, Canada

    J. O. Bustamante

  3. Johns Hopkins University, Wilmer Eye Institute, 21287, Baltimore, MD

    A. Liepins & R. A. Prendergast

  4. Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892, Bethesda, MD

    J. A. Hanover

  5. Physiologisches Institut der Universität Würzburg, D 97070, Würzburg, Germany

    H. Oberleithner

Authors
  1. J. O. Bustamante
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Liepins
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. A. Prendergast
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. A. Hanover
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H. Oberleithner
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Supported by grants from the American Heart Association, Maryland Affiliate, to JOB, from the Medical Research Council of Canada to AL, from Research to Prevent Blindness to RAP, from National Institutes of Health Intramural Funding to JAH, and from the Deutsche Forschungsgemeinschaft, SFB 176 (A 6) to HO. The authors thank Peggy Kopps of Promega for her assistance in the calculation of TBP concentration and in finding biochemical applications where the transcription factor displays equally strong stabilizing effects.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bustamante, J.O., Liepins, A., Prendergast, R.A. et al. Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex. J. Membarin Biol. 146, 263–272 (1995). https://doi.org/10.1007/BF00233946

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00233946

Key words

Search

Navigation