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PMID: 7828583 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Determination of heat-shock transcription factor 2 stoichiometry at looped DNA complexes using scanning force microscopy.

The EMBO journal ·Vol. 14 ·No. 1 ·1995-01-03 ·Pages 117-23

Wyman C, Grotkopp E, Bustamante C, Nelson HC

Abstract

Gene activation frequently requires an array of proteins bound to sites distal to the transcription start site. The assembly of these protein-bound sites into specialized nucleoprotein complexes is a prerequisite for transcriptional activation. Structural analysis of these higher order complexes will provide crucial information for understanding the mechanisms of gene activation. We have used both electron microscopy and scanning force microscopy to elucidate the structure of complexes formed between DNA and heat-shock transcription factor (HSF) 2, a human heat-shock transcriptional activator that binds DNA as a trimer. Electron microscopy reveals that HSF2 will bring together distant DNA sites to create a loop. We show that this association requires only the DNA binding and trimerization domains of HSF2. Metal shadowing techniques used for electron microscopy obscure details of these nucleoprotein structures. Greatly increased resolution was achieved by directly imaging the complexes in the scanning force microscope, which reveals that at least two trimers are required for the association of HSF2-bound DNA sites.

MeSH Terms
DNA/metabolism,ultrastructure Heat-Shock Proteins/genetics,metabolism,ultrastructure Humans Microscopy, Atomic Force/methods Microscopy, Electron/methods Nucleic Acid Conformation Protein Binding Protein Conformation Recombinant Proteins/metabolism,ultrastructure Transcription Factors/genetics,metabolism,ultrastructure
Chemicals
Heat-Shock Proteins Recombinant Proteins Transcription Factors HSF2 protein, human DNA
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Wyman C
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
Grotkopp E
Bustamante C
Nelson H C
References (43)
43 references, click to expand
  1. Mouse heat shock transcription factors 1 and 2 prefer a trimeric binding site but interact differently with the HSP70 heat shock element.
    Mol Cell Biol. 1993 Jun;13(6):3370-83 PMID: 8497256
  2. Use of polylysine for adsorption of nuclei acids and enzymes to electron microscope specimen films.
    Proc Natl Acad Sci U S A. 1977 Jun;74(6):2311-5 PMID: 329278
  3. Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites.
    Mol Cell Biol. 1993 Nov;13(11):6941-56 PMID: 8105373
  4. Constitutive binding of yeast heat shock factor to DNA in vivo.
    Mol Cell Biol. 1988 Nov;8(11):5040-2 PMID: 3062378
  5. Genomic footprinting of the yeast HSP82 promoter reveals marked distortion of the DNA helix and constitutive occupancy of heat shock and TATA elements.
    J Mol Biol. 1990 Dec 5;216(3):611-31 PMID: 2175361
  6. Characterization of a novel chicken heat shock transcription factor, heat shock factor 3, suggests a new regulatory pathway.
    Mol Cell Biol. 1993 Apr;13(4):1983-97 PMID: 8455593
  7. Activation of the Drosophila hsp27 promoter by heat shock and by ecdysone involves independent and remote regulatory sequences.
    EMBO J. 1986 Jul;5(7):1653-8 PMID: 16453691
  8. Human heat shock factors 1 and 2 are differentially activated and can synergistically induce hsp70 gene transcription.
    Mol Cell Biol. 1994 Mar;14(3):2087-99 PMID: 8114740
  9. Protein traffic on the heat shock promoter: parking, stalling, and trucking along.
    Cell. 1993 Jul 16;74(1):1-4 PMID: 8334697
  10. Germline transformation used to define key features of heat-shock response elements.
    Science. 1988 Mar 4;239(4844):1139-42 PMID: 3125608
  11. Circular DNA molecules imaged in air by scanning force microscopy.
    Biochemistry. 1992 Jan 14;31(1):22-6 PMID: 1310032
  12. Stabilization of the EBNA1 protein on the Epstein-Barr virus latent origin of DNA replication by a DNA looping mechanism.
    J Biol Chem. 1994 Jan 14;269(2):1057-62 PMID: 8288561
  13. Purification and characterization of a heat-shock element binding protein from yeast.
    EMBO J. 1987 Oct;6(10):3035-41 PMID: 3319580
  14. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp70 gene and when linked to a heterologous promoter.
    Cell. 1986 Jun 6;45(5):753-60 PMID: 3085957
  15. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation.
    Cell. 1988 Sep 9;54(6):855-64 PMID: 3044613
  16. A germline transformation analysis reveals flexibility in the organization of heat shock consensus elements.
    Nucleic Acids Res. 1987 Apr 10;15(7):2971-88 PMID: 3562243
  17. Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat shock transcription factor.
    J Biol Chem. 1993 Nov 25;268(33):25229-38 PMID: 8227088
  18. Periodic interactions of heat shock transcriptional elements.
    Nature. 1988 Apr 28;332(6167):856-8 PMID: 2833709
  19. Cooperative binding of heat shock transcription factor to the Hsp70 promoter in vivo and in vitro.
    J Biol Chem. 1994 Feb 18;269(7):4804-11 PMID: 8106450
  20. Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation.
    Cell. 1990 Nov 30;63(5):1085-97 PMID: 2257625
  21. Yeast heat shock transcription factor contains a flexible linker between the DNA-binding and trimerization domains. Implications for DNA binding by trimeric proteins.
    J Biol Chem. 1994 Apr 29;269(17):12475-81 PMID: 8175654
  22. A conserved heptapeptide restrains the activity of the yeast heat shock transcription factor.
    EMBO J. 1991 Feb;10(2):369-75 PMID: 1899375
  23. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit.
    Cell. 1989 Dec 1;59(5):797-806 PMID: 2590940
  24. The activation domain of the bovine papillomavirus E2 protein mediates association of DNA-bound dimers to form DNA loops.
    Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3204-8 PMID: 1849647
  25. Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress.
    Mol Cell Biol. 1993 Mar;13(3):1392-407 PMID: 8441385
  26. Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF.
    EMBO J. 1990 Dec;9(13):4495-501 PMID: 2148291
  27. Use of T7 RNA polymerase to direct expression of cloned genes.
    Methods Enzymol. 1990;185:60-89 PMID: 2199796
  28. Isolation of a cDNA for HSF2: evidence for two heat shock factor genes in humans.
    Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6911-5 PMID: 1871106
  29. Interactions between DNA-bound trimers of the yeast heat shock factor.
    Mol Cell Biol. 1994 Jan;14(1):501-8 PMID: 8264619
  30. Protein/DNA architecture of the DNase I hypersensitive region of the Drosophila hsp26 promoter.
    EMBO J. 1988 Jul;7(7):2191-201 PMID: 2901349
  31. DNA looping and unlooping by AraC protein.
    Science. 1990 Oct 26;250(4980):528-32 PMID: 2237403
  32. Trimerization of a yeast transcriptional activator via a coiled-coil motif.
    Cell. 1989 Dec 1;59(5):807-13 PMID: 2686840
  33. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor.
    Cell. 1988 Sep 9;54(6):841-53 PMID: 3044612
  34. A distal heat shock element promotes the rapid response to heat shock of the HSP26 gene in the yeast Saccharomyces cerevisiae.
    J Biol Chem. 1993 Apr 5;268(10):7442-8 PMID: 8463277
  35. Molecular cloning and expression of a human heat shock factor, HSF1.
    Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6906-10 PMID: 1871105
  36. Activation of human heat shock genes is accompanied by oligomerization, modification, and rapid translocation of heat shock transcription factor HSF1.
    Mol Cell Biol. 1993 Apr;13(4):2486-96 PMID: 8455624
  37. DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1.
    Genes Dev. 1991 May;5(5):820-6 PMID: 1851121
  38. Association of DNA-bound progesterone receptors.
    Nature. 1987 Sep 3-9;329(6134):79-81 PMID: 3041228
  39. Key features of heat shock regulatory elements.
    Mol Cell Biol. 1988 Sep;8(9):3761-9 PMID: 3146692
  40. Sequences required for in vitro transcriptional activation of a Drosophila hsp 70 gene.
    Cell. 1985 Sep;42(2):527-37 PMID: 4028160
  41. Activation of Drosophila heat shock factor: conformational change associated with a monomer-to-trimer transition.
    Mol Cell Biol. 1993 Jun;13(6):3481-6 PMID: 8497263
  42. Cooperative binding of Drosophila heat shock factor to arrays of a conserved 5 bp unit.
    Cell. 1991 Feb 8;64(3):585-93 PMID: 1899357
  43. Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability.
    Genes Dev. 1991 Oct;5(10):1902-11 PMID: 1717345
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1995-01-03
Pages
117-23
Language
English
Region
England
NLM ID
8208664
PMCID
PMC398058
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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