Skip to main content
SpringerLink
Log in

Lac repressor — Lac operator complexes

Solution X-ray scattering and electrophoretic studies

  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

Complexes between the Lac repressor and a small DNA operator fragment (29 base pairs) were investigated using polyacrylamide gel electrophoresis and solution X-ray scattering. Titration of the DNA fragment with the repressor, followed by gel electrophoresis showed that only two types of complexes are formed with repressor/operator ratios of 0.5 and 2. Radii of gyration and forward scattered intensities were obtained from Guinier plots for repressor/operator ratios ranging from 0.3 to 2. They demonstrated that the first complex contains one repressor and two operators, whereas the second one contains four repressors and two operators. Mixing operator and repressor in equimolar concentrations leads to a mixture of both complexes. A possible model for the four repressor/two operator complex is proposed.

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

  • Barbier B, Charlier M, Maurizot J-C (1984) Photochemical cross-linking of Lac repressor to nonoperator 5-bromouracil-substituted DNA. Biochemistry 23:2933–2939

    Article  CAS  Google Scholar 

  • Barkley MD, Riggs AD, Jobe A, Bourgeois S (1975) Interaction of effecting ligands with Lac repressor and repressor-operator complex. Biochemistry 14:1700–1712

    Article  CAS  Google Scholar 

  • Bolivar F, Betlach MC, Heyneker HL, Shine J, Rodriguez RL, Boyer HW (1977) Origin of replication of pBr345 plasmid DNA. Proc Natl Acad Sci USA 74:5265–5269

    Article  CAS  Google Scholar 

  • Bourgeois S, Pfahl M (1976) Repressors. Adv Protein Chem 30:1–99

    Article  CAS  Google Scholar 

  • Bück F, Hahn K-D, Zemann W, Rüterjans H, Sadler JR, Beyreuther K, Kaptein R, Scheek R, Hull WE (1983) NMR study of the interaction between the Lac repressor and the Lac operator. Eur J Biochem 132:321–327

    Article  Google Scholar 

  • Butler AP, Revzin A, von Hippel PH (1977) Molecular parameters characterizing the interaction of Escherichia coli Lac repressor with non-operator DNA and inducer. Biochemistry 16:4757–4768

    Article  CAS  Google Scholar 

  • Charlier M, Maurizot J-C (1983) Non-specific binding of Lac repressor to DNA. I. An absorption and circular dichroism study. Biophys Chem 18:303–311

    Article  CAS  Google Scholar 

  • Charlier M, Maurizot J-C, Zaccai G (1980) Neutron scattering studies of Lac repressor. Nature 286:423–425

    Article  CAS  Google Scholar 

  • Charlier M, Maurizot J-C, Zaccai G (1981) Neutron scattering studies of Lac repressor: A low resolution model. J Mol Biol 153:177–182

    Article  CAS  Google Scholar 

  • Charlier M, Maurizot J-C, Zaccai G (1983) Non-specific binding of Lac repressor to DNA. II. A small-angle neutron scattering study. Biophys Chem 18:313–322

    Article  CAS  Google Scholar 

  • Chothia C (1975) Structural invariants in protein folding. Nature 254:304–308

    Article  CAS  Google Scholar 

  • Crothers DM, Fried M (1983) Transmission of long range effects in DNA. Cold Spring Harbor Symp Quant Biol 47:263–269

    Article  Google Scholar 

  • Culard F, Maurizot J-C (1981) Lac repressor — Lac operator interaction. Circular dichroism study. Nucl Acids Res 9:5175–5184

    Article  CAS  Google Scholar 

  • Culard F, Maurizot J-C (1982) Binding of Lac repressor induces different conformational changes on operator and non-operator DNAs. FEBS Lett 146:153–156

    Article  CAS  Google Scholar 

  • Culard F, Schnarr M, Maurizot J-C (1982) Interaction between the Lac operator and the Lac repressor headpiece: fluorescence and circular dichroism studies. EMBO J 1:1405–1409

    Article  CAS  Google Scholar 

  • Dunaway M, Manly SP, Matthews KS (1980) Model for lactose repressor protein and its interaction with ligands. Proc Natl Acad Sci USA 77:7181–7185

    Article  CAS  Google Scholar 

  • Fried M, Crothers DM (1981) Equilibria and kinetics of Lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucl Acids Res 9:6505–6525

    Article  CAS  Google Scholar 

  • Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: Application to components of the Escherichia coli lactose operon regulatory system. Nucl Acids Res 9:3047–3060

    Article  CAS  Google Scholar 

  • Gilbert W, Maxam A (1973) The nucleotide sequence of the Lac operator. Proc Natl Acad Sci USA 70:3581–3584

    Article  CAS  Google Scholar 

  • Irani MH, Orosz L, Adhya S (1983) A control element within a structural gene: The gal operon of Escherichia coli. Cell 32:783–788

    Article  CAS  Google Scholar 

  • Jacrot B (1976) The study of biological structures by neutron scattering from solution. Rep Prog Phys 39:911–953

    Article  CAS  Google Scholar 

  • Jovin T, Geisler N, Weber K (1977) Amino-terminal fragments of Escherichia coli Lac repressor binds to DNA. Nature 269:668–672

    Article  CAS  Google Scholar 

  • Kallai OB, Rosenberg JM, Kopka ML, Takano T, Dickerson RE, Kan J, Riggs AD (1980) Large-scale purification of two forms of active Lac operator from plasmids. Biochim Biophys Acta 606:113–124

    Article  CAS  Google Scholar 

  • Kania J, Müller-Hill B (1977) Construction, isolation and implications of repressor-galactosidase. β-galactosidase hybrid molecules. Eur J Biochem 79:381–386

    Article  CAS  Google Scholar 

  • Kaptein R, Zuiderweg ERP, Scheek RM, Boelens R, Gunsteren WF (1985) A protein structure from nuclear magnetic resonance data. Lac repressor headpiece. J Mol Biol 182:179–182

    Article  CAS  Google Scholar 

  • Koch MHJ, Stuhrmann HB, Tardieu A, Vachette P (1982) Small angle X-ray scattering of solutions. In: Stuhrmann HB (ed) Uses of synchrotron radiation in biology. Academic Press, New York, pp 223–253

    Google Scholar 

  • Kolb A, Busby S, Herbert M, Kotlarz D, Buc H (1983) Comparison of the binding sites for the Escherichia coli cAMP receptor protein at the lactose and galactose promoters. EMBO J 2:217–222

    Article  CAS  Google Scholar 

  • Lillis M, Nick H, Lu P, Sadler J, Caruthers MH (1982) Lac operon DNA: isolation and trimming for NMR spectroscopy. Anal Biochem 120:52–58

    Article  CAS  Google Scholar 

  • Lin SY, Riggs AD (1972) Lac repressor binding to nonoperator DNA: Detailed studies and a comparison of equilibrium and rate competition model. J Mol Biol 72:671–690

    Article  CAS  Google Scholar 

  • Lin S-Y, Riggs AD (1975) The general affinity of Lac repressor for E. coli DNA: Implications for gene regulation in procaryotes and eucaryotes. Cell 4:107–111

    Article  CAS  Google Scholar 

  • Luzzati V, Tardieu A (1980) Recent developments in solution X-ray scattering. Annu Rev Biophys Bioeng 9:1–29

    Article  CAS  Google Scholar 

  • Manly SP, Bennett GN, Matthews KS (1983a) Enzymatic digestion of operator DNA in the presence of the Lac repressor tryptic core. J Mol Biol 179:335–350

    Article  Google Scholar 

  • Manly SP, Bennett GN, Matthews KS (1983b) Perturbation of Lac operator DNA modification by tryptic core protein from Lac repressor. Proc Natl Acad Sci USA 80:6219–6223

    Article  CAS  Google Scholar 

  • Matthews KS (1979) Tryptic core protein of lactose repressor binds operator DNA. J Biol Chem 254:3348–3353

    CAS  PubMed  Google Scholar 

  • Maurizot J-C, Charlier M, Helene C (1974) Lac repressor binding to poly(dAT). Conformational changes. Biochem Biophys Res Commun 60:951–957

    Article  CAS  Google Scholar 

  • Maxam A, Gibert W (1980) Sequencing end-labelded DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560

    Article  CAS  Google Scholar 

  • McKay DB, Pickover CA, Steitz TA (1982) Escherichia coli Lac repressor is elongated with its operator DNA binding domains located at both ends. J Mol Biol 156:175–183

    Article  CAS  Google Scholar 

  • Müller-Hill B (1975) Lac repressor and Lac operator. Prog Biophys Mol Biol 30:227–252

    Article  Google Scholar 

  • Nick H, Arndt K, Boschelli F, Jarema MAC, Lillis M, Sadler J, Caruthers M, Lu P (1982a) Lac repressor — Lac operator interaction: NMR observations. Proc Natl Acad Sci USA 79:218–222

    Article  CAS  Google Scholar 

  • Nick H, Arndt K, Boschelli F, Jarema MAC, Lillis M, Sommer H, Lu P, Sadler J (1982b) Repressor-operator interaction in the Lac operon. II. Observations at the tyrosines and tryptophans. J Mol Biol 161:417–438

    Article  CAS  Google Scholar 

  • Ogata RT, Gilbert W (1979) DNA binding site of Lac repressor probed by dimehtylsulfate methylation of Lac operator. J Mol Biol 132:709–728

    Article  CAS  Google Scholar 

  • O'Gorman RB, Dunaway M, Matthews KS (1980a) DNA binding characteristics of lactose repressor and the trypsin resistant core repressor. J Biol Chem 255:10100–10106

    CAS  PubMed  Google Scholar 

  • O'Gorman RB, Rosenberg JM, Kallai OB, Dickerson RE, Itakura K, Riggs AD, Matthews KS (1980b) Equilibrium binding of inducer to Lac repressor-operator DNA complex. J Biol Chem 255:10107–10114

    CAS  PubMed  Google Scholar 

  • Platt T, Files JG, Weber K (1973) Lac repressor. Specific proteolytic destruction of the NH2-terminal region and loss of the deoxyribonucleic acid-binding activity. J Biol Chem 248:110–121

    CAS  PubMed  Google Scholar 

  • Record MT, de Haseth PL, Lohman TM (1977) Interpretation of monovalent and divalent cation effect on the lac repressor-operator interaction. Biochemistry 16:4791–4796

    Article  CAS  Google Scholar 

  • Revzin A, von Hippel PH (1977) Direct measurement of association constants for the binding of Escherichia coli Lac repressor to non-operator DNA. Biochemistry 16:4769–4776

    Article  CAS  Google Scholar 

  • Rosenberg JM, Kallai OB, Kopka ML, Dickerson RE, Riggs AD (1977) Lac repressor purification without inactivation of DNA binding activity. Nucl Acids Res 4:567–572

    Article  CAS  Google Scholar 

  • Scheek RM, Zuiderweg ERP, Klappe KJM, van Boom JH, Kaptein R, Rüterjans H, Beyreuther K (1983) Lac repressor headpiece binds specifically to half of the Lac operator: A nuclear magnetic resonance study. Biochemistry 22:228–235

    Article  CAS  Google Scholar 

  • Steitz TA, Richmond TJ, Wise D, Engelman D (1974) The Lac repressor protein: Molecular shape, subunits structure, and proposed model for operator interaction based on structural studies on microcrystals. Proc Natl Acad Sci USA 71:593–597

    Article  CAS  Google Scholar 

  • von Wilcken-Bergmann B, Müller-Hill B (1982) Sequence of galR gene indicates a common evolutionary origin of lac and gal repressor in Escherichia coli. Proc Natl Acad Sci USA 79:2427–2431

    Article  Google Scholar 

  • Weber K, Osborn M (1975) Protein and sodium dodecyl sulfate: Molecular weight determination on polyacrylamide gels and related procedures. In: Neurath H, Hill RL (eds) The proteins, vol 1, 3rd ed. Academic Press, New York London, pp 179–223

    Chapter  Google Scholar 

  • Winter RB, von Hippel PH (1981) Diffusion-driven mechanism of protein translocation on nucleic acids. 2. The Escherichia coli repressor-operator interaction: Equilibrium measurements. Biochemistry 20:6948–6960

    Article  CAS  Google Scholar 

  • Zamyatnin AA (1972) Protein volume in solution. Prog Biophys Mol Biol 24:107–123

    Article  CAS  Google Scholar 

  • Zuiderweg ERP, Billeter M, Boelens R, Scheek RM, Wüthrich K, Kaptein R (1984) Spatial arrangement of the three α-helices in the solution conformation of E. coli Lac repressor DNA binding domain. FEBS Lett 174:243–247

    Article  CAS  Google Scholar 

  • Zuiderweg ERP, Kaptein R, Wüthrich K (1983) Secondary structure of the Lac repressor DNA binding domain by two dimensional 1H nuclear magnetic resonance in solution. Proc Natl Acad Sci USA 80:5837–5841

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centre de Biophysique Moléculaire, C.N.R.S., 1A, avenue de la Recherche Scientifique, F-45071, Orleans Cedex 2, France

    F. Culard, M. Charlier & J. -C. Maurizot

  2. Centre de Génétique Moléculaire, C.N.R.S., F-91190, Gif-sur-Yvette, France

    A. Tardieu

Authors
  1. F. Culard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Charlier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. -C. Maurizot
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Tardieu
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Culard, F., Charlier, M., Maurizot, J.C. et al. Lac repressor — Lac operator complexes. Eur Biophys J 14, 169–178 (1987). https://doi.org/10.1007/BF00253842

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00253842

Key words

Search

Navigation