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. 1996 Apr 2;93(7):2920–2925. doi: 10.1073/pnas.93.7.2920

In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.

T Sera 1, P G Schultz 1
PMCID: PMC39735  PMID: 8610143

Abstract

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Choo Y., Klug A. Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11163–11167. doi: 10.1073/pnas.91.23.11163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Elledge S. J., Davis R. W. Position and density effects on repression by stationary and mobile DNA-binding proteins. Genes Dev. 1989 Feb;3(2):185–197. doi: 10.1101/gad.3.2.185. [DOI] [PubMed] [Google Scholar]
  3. Elledge S. J., Sugiono P., Guarente L., Davis R. W. Genetic selection for genes encoding sequence-specific DNA-binding proteins. Proc Natl Acad Sci U S A. 1989 May;86(10):3689–3693. doi: 10.1073/pnas.86.10.3689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  5. Glover J. N., Harrison S. C. Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature. 1995 Jan 19;373(6511):257–261. doi: 10.1038/373257a0. [DOI] [PubMed] [Google Scholar]
  6. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  7. Jamieson A. C., Kim S. H., Wells J. A. In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry. 1994 May 17;33(19):5689–5695. doi: 10.1021/bi00185a004. [DOI] [PubMed] [Google Scholar]
  8. Jarvis T. C., Ring D. M., Daube S. S., von Hippel P. H. "Macromolecular crowding": thermodynamic consequences for protein-protein interactions within the T4 DNA replication complex. J Biol Chem. 1990 Sep 5;265(25):15160–15167. [PubMed] [Google Scholar]
  9. Johnson P. F. Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference. Mol Cell Biol. 1993 Nov;13(11):6919–6930. doi: 10.1128/mcb.13.11.6919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. König P., Richmond T. J. The X-ray structure of the GCN4-bZIP bound to ATF/CREB site DNA shows the complex depends on DNA flexibility. J Mol Biol. 1993 Sep 5;233(1):139–154. doi: 10.1006/jmbi.1993.1490. [DOI] [PubMed] [Google Scholar]
  11. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  12. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  13. Mossing M. C., Record M. T., Jr Thermodynamic origins of specificity in the lac repressor-operator interaction. Adaptability in the recognition of mutant operator sites. J Mol Biol. 1985 Nov 20;186(2):295–305. doi: 10.1016/0022-2836(85)90106-8. [DOI] [PubMed] [Google Scholar]
  14. Nelson H. C., Sauer R. T. Lambda repressor mutations that increase the affinity and specificity of operator binding. Cell. 1985 Sep;42(2):549–558. doi: 10.1016/0092-8674(85)90112-6. [DOI] [PubMed] [Google Scholar]
  15. Paolella D. N., Palmer C. R., Schepartz A. DNA targets for certain bZIP proteins distinguished by an intrinsic bend. Science. 1994 May 20;264(5162):1130–1133. doi: 10.1126/science.8178171. [DOI] [PubMed] [Google Scholar]
  16. Pomerantz J. L., Sharp P. A., Pabo C. O. Structure-based design of transcription factors. Science. 1995 Jan 6;267(5194):93–96. doi: 10.1126/science.7809612. [DOI] [PubMed] [Google Scholar]
  17. Rebar E. J., Pabo C. O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science. 1994 Feb 4;263(5147):671–673. doi: 10.1126/science.8303274. [DOI] [PubMed] [Google Scholar]
  18. Rhodes D., Klug A. Zinc fingers. Sci Am. 1993 Feb;268(2):56-9, 62-5. doi: 10.1038/scientificamerican0293-56. [DOI] [PubMed] [Google Scholar]
  19. Rørth P. Specification of C/EBP function during Drosophila development by the bZIP basic region. Science. 1994 Dec 16;266(5192):1878–1881. doi: 10.1126/science.7997882. [DOI] [PubMed] [Google Scholar]
  20. Suckow M., Madan A., Kisters-Woike B., von Wilcken-Bergmann B., Müller-Hill B. Creating new DNA binding specificities in the yeast transcriptional activator GCN4 by combining selected amino acid substitutions. Nucleic Acids Res. 1994 Jun 25;22(12):2198–2208. doi: 10.1093/nar/22.12.2198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Talanian R. V., McKnight C. J., Kim P. S. Sequence-specific DNA binding by a short peptide dimer. Science. 1990 Aug 17;249(4970):769–771. doi: 10.1126/science.2389142. [DOI] [PubMed] [Google Scholar]
  22. Wharton R. P., Ptashne M. Changing the binding specificity of a repressor by redesigning an alpha-helix. Nature. 1985 Aug 15;316(6029):601–605. doi: 10.1038/316601a0. [DOI] [PubMed] [Google Scholar]
  23. Yu M., Ojwang J., Yamada O., Hampel A., Rapapport J., Looney D., Wong-Staal F. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6340–6344. doi: 10.1073/pnas.90.13.6340. [DOI] [PMC free article] [PubMed] [Google Scholar]

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