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. 1995 Dec 15;14(24):6292–6300. doi: 10.1002/j.1460-2075.1995.tb00319.x

Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target.

S W Yang 1, H A Nash 1
PMCID: PMC394753  PMID: 8557048

Abstract

We have quantitatively evaluated the affinity of a set of target sites for the integration host factor (IHF) protein of Escherichia coli by their performance as competitors in an electrophoretic mobility shift assay. We also determined how well each of these sites is filled by IHF in vivo. The data show that several natural sites have an affinity not much greater than that required for intracellular occupancy. The data also indicate that very little of the IHF in a cell is present as free protein available for binding, suggesting that binding to non-specific targets dominates the operation of this system. The correlation between in vitro affinity and in vivo occupancy provides a ready means to assess the likely physiological significance of putative IHF sites. It also provides a general method to assess the importance of non-specific interactions by DNA binding proteins inside a cell.

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Selected References

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  1. Baker R. E., Gabrielsen O., Hall B. D. Effects of tRNATyr point mutations on the binding of yeast RNA polymerase III transcription factor C. J Biol Chem. 1986 Apr 25;261(12):5275–5282. [PubMed] [Google Scholar]
  2. Boccard F., Prentki P. Specific interaction of IHF with RIBs, a class of bacterial repetitive DNA elements located at the 3' end of transcription units. EMBO J. 1993 Dec 15;12(13):5019–5027. doi: 10.1002/j.1460-2075.1993.tb06195.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boffini A., Prentki P. Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis. Nucleic Acids Res. 1991 Apr 11;19(7):1369–1374. doi: 10.1093/nar/19.7.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bushman W., Thompson J. F., Vargas L., Landy A. Control of directionality in lambda site specific recombination. Science. 1985 Nov 22;230(4728):906–911. doi: 10.1126/science.2932798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  6. Cook D. I., Revzin A. Intracellular location of catabolite activator protein of Escherichia coli. J Bacteriol. 1980 Mar;141(3):1279–1283. doi: 10.1128/jb.141.3.1279-1283.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craig N. L., Nash H. A. E. coli integration host factor binds to specific sites in DNA. Cell. 1984 Dec;39(3 Pt 2):707–716. doi: 10.1016/0092-8674(84)90478-1. [DOI] [PubMed] [Google Scholar]
  8. Ditto M. D., Roberts D., Weisberg R. A. Growth phase variation of integration host factor level in Escherichia coli. J Bacteriol. 1994 Jun;176(12):3738–3748. doi: 10.1128/jb.176.12.3738-3748.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fried M. G., Crothers D. M. Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J Mol Biol. 1984 Jan 25;172(3):263–282. doi: 10.1016/s0022-2836(84)80026-1. [DOI] [PubMed] [Google Scholar]
  10. Friedman D. I. Integration host factor: a protein for all reasons. Cell. 1988 Nov 18;55(4):545–554. doi: 10.1016/0092-8674(88)90213-9. [DOI] [PubMed] [Google Scholar]
  11. Gardner J. F., Nash H. A. Role of Escherichia coli IHF protein in lambda site-specific recombination. A mutational analysis of binding sites. J Mol Biol. 1986 Sep 20;191(2):181–189. doi: 10.1016/0022-2836(86)90255-x. [DOI] [PubMed] [Google Scholar]
  12. Giladi H., Goldenberg D., Koby S., Oppenheim A. B. Enhanced activity of the bacteriophage lambda PL promoter at low temperature. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2184–2188. doi: 10.1073/pnas.92.6.2184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodman S. D., Nash H. A. Functional replacement of a protein-induced bend in a DNA recombination site. Nature. 1989 Sep 21;341(6239):251–254. doi: 10.1038/341251a0. [DOI] [PubMed] [Google Scholar]
  14. Goodrich J. A., Schwartz M. L., McClure W. R. Searching for and predicting the activity of sites for DNA binding proteins: compilation and analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 1990 Sep 11;18(17):4993–5000. doi: 10.1093/nar/18.17.4993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hales L. M., Gumport R. I., Gardner J. F. Determining the DNA sequence elements required for binding integration host factor to two different target sites. J Bacteriol. 1994 May;176(10):2999–3006. doi: 10.1128/jb.176.10.2999-3006.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kur J., Hasan N., Szybalski W. Physical and biological consequences of interactions between integration host factor (IHF) and coliphage lambda late p'R promoter and its mutants. Gene. 1989 Sep 1;81(1):1–15. doi: 10.1016/0378-1119(89)90331-4. [DOI] [PubMed] [Google Scholar]
  17. Lin S., Riggs A. D. The general affinity of lac repressor for E. coli DNA: implications for gene regulation in procaryotes and eucaryotes. Cell. 1975 Feb;4(2):107–111. doi: 10.1016/0092-8674(75)90116-6. [DOI] [PubMed] [Google Scholar]
  18. Linden J. Calculating the dissociation constant of an unlabeled compound from the concentration required to displace radiolabel binding by 50%. J Cyclic Nucleotide Res. 1982;8(3):163–172. [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  20. Molina-López J. A., Govantes F., Santero E. Geometry of the process of transcription activation at the sigma 54-dependent nifH promoter of Klebsiella pneumoniae. J Biol Chem. 1994 Oct 14;269(41):25419–25425. [PubMed] [Google Scholar]
  21. Nash H. A., Robertson C. A. Heteroduplex substrates for bacteriophage lambda site-specific recombination: cleavage and strand transfer products. EMBO J. 1989 Nov;8(11):3523–3533. doi: 10.1002/j.1460-2075.1989.tb08518.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oberto J., Drlica K., Rouvière-Yaniv J. Histones, HMG, HU, IHF: Même combat. Biochimie. 1994;76(10-11):901–908. doi: 10.1016/0300-9084(94)90014-0. [DOI] [PubMed] [Google Scholar]
  23. Oppenheim A. B., Rudd K. E., Mendelson I., Teff D. Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli. Mol Microbiol. 1993 Oct;10(1):113–122. doi: 10.1111/j.1365-2958.1993.tb00908.x. [DOI] [PubMed] [Google Scholar]
  24. Pagel J. M., Winkelman J. W., Adams C. W., Hatfield G. W. DNA topology-mediated regulation of transcription initiation from the tandem promoters of the ilvGMEDA operon of Escherichia coli. J Mol Biol. 1992 Apr 20;224(4):919–935. doi: 10.1016/0022-2836(92)90460-2. [DOI] [PubMed] [Google Scholar]
  25. Pérez-Martín J., Timmis K. N., de Lorenzo V. Co-regulation by bent DNA. Functional substitutions of the integration host factor site at sigma 54-dependent promoter Pu of the upper-TOL operon by intrinsically curved sequences. J Biol Chem. 1994 Sep 9;269(36):22657–22662. [PubMed] [Google Scholar]
  26. Richet E., Abcarian P., Nash H. A. The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. Cell. 1986 Sep 26;46(7):1011–1021. doi: 10.1016/0092-8674(86)90700-2. [DOI] [PubMed] [Google Scholar]
  27. Ruusala T., Crothers D. M. Sliding and intermolecular transfer of the lac repressor: kinetic perturbation of a reaction intermediate by a distant DNA sequence. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):4903–4907. doi: 10.1073/pnas.89.11.4903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sasse-Dwight S., Gralla J. D. Footprinting protein-DNA complexes in vivo. Methods Enzymol. 1991;208:146–168. doi: 10.1016/0076-6879(91)08012-7. [DOI] [PubMed] [Google Scholar]
  29. Sawadogo M. Multiple forms of the human gene-specific transcription factor USF. II. DNA binding properties and transcriptional activity of the purified HeLa USF. J Biol Chem. 1988 Aug 25;263(24):11994–12001. [PubMed] [Google Scholar]
  30. Segall A. M., Goodman S. D., Nash H. A. Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins. EMBO J. 1994 Oct 3;13(19):4536–4548. doi: 10.1002/j.1460-2075.1994.tb06775.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stickle D. F., Vossen K. M., Riley D. A., Fried M. G. Free DNA concentration in E. coli estimated by an analysis of competition for DNA binding proteins. J Theor Biol. 1994 May 7;168(1):1–12. doi: 10.1006/jtbi.1994.1082. [DOI] [PubMed] [Google Scholar]
  32. Thompson J. F., Moitoso de Vargas L., Koch C., Kahmann R., Landy A. Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway. Cell. 1987 Sep 11;50(6):901–908. doi: 10.1016/0092-8674(87)90516-2. [DOI] [PubMed] [Google Scholar]
  33. Wang S., Cosstick R., Gardner J. F., Gumport R. I. The specific binding of Escherichia coli integration host factor involves both major and minor grooves of DNA. Biochemistry. 1995 Oct 10;34(40):13082–13090. doi: 10.1021/bi00040a020. [DOI] [PubMed] [Google Scholar]
  34. Xin W., Feiss M. Function of IHF in lambda DNA packaging. I. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB. J Mol Biol. 1993 Mar 20;230(2):492–504. doi: 10.1006/jmbi.1993.1166. [DOI] [PubMed] [Google Scholar]
  35. Yang C. C., Nash H. A. The interaction of E. coli IHF protein with its specific binding sites. Cell. 1989 Jun 2;57(5):869–880. doi: 10.1016/0092-8674(89)90801-5. [DOI] [PubMed] [Google Scholar]
  36. Zimmerman S. B., Trach S. O. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol. 1991 Dec 5;222(3):599–620. doi: 10.1016/0022-2836(91)90499-v. [DOI] [PubMed] [Google Scholar]
  37. Zulianello L., van Ulsen P., van de Putte P., Goosen N. Participation of the flank regions of the integration host factor protein in the specificity and stability of DNA binding. J Biol Chem. 1995 Jul 28;270(30):17902–17907. doi: 10.1074/jbc.270.30.17902. [DOI] [PubMed] [Google Scholar]
  38. van Rijn P. A., van de Putte P., Goosen N. Analysis of the IHF binding site in the regulatory region of bacteriophage Mu. Nucleic Acids Res. 1991 Jun 11;19(11):2825–2834. doi: 10.1093/nar/19.11.2825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. von Hippel P. H., Revzin A., Gross C. A., Wang A. C. Non-specific DNA binding of genome regulating proteins as a biological control mechanism: I. The lac operon: equilibrium aspects. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4808–4812. doi: 10.1073/pnas.71.12.4808. [DOI] [PMC free article] [PubMed] [Google Scholar]

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