Abstract
Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA - 6 base-pairs - NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10 to 50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Greisman, H.A. and Pabo, C.O. (1997) A general strategy for selecting high-affinity zinc finger proteins for diverse DNA sites, Science, 275, 657–661.
Kim, J.-S. and Pabo, C.O. (1998) Getting a handhold on DNA: Design of poly-zinc finger proteins with femtomolar dissociation constants, Proc. Natl. Acad. Sci. USA, 95, 2812–2817.
Liu, Q., Segal, D.J., Ghiara, J.B. and Barbas, C.F., III. (1997) Design of polydactyl zinc-finger proteins for unique addressing within complex genomes, Proc. Natl.Acad. Sci. USA, 94, 5525–5530.
Choo, Y., Castellanos, A., Garcia-Hernandez, B., Sanchez-Garcia, I. and Klug, A. (1997) Promoter-specific activation of gene expression directed by bacteriophageselected zinc fingers, J. Mol. Biol 273, 525–532.
Kim, J.-S. and Pabo, C.O. (1997) Transcriptional repression by zinc finger peptides. Exploring the potential for applications in gene therapy, J. Biol. Chem 272, 29795–29800.
Berg, J.M. (1997) Letting your fingers do the walking, Nature Biotech 15, 323.
Choo, Y. and Klug, A. (1995) Designing DNA-binding proteins on the surface of filamentous phage, Curr. Opin. Biotechnol 6, 431–436.
Rebar, E.J., Greisman, H.A. and Pabo, C.O. (1996) Phage display methods for selecting zinc finger proteins with novel DNA-binding specificities, Methods Enzymol 267, 129–149.
Berg, J.M. and Shi, Y. (1996) Galvanization of biology: a growing appreciation for the roles of zinc, Science, 271, 1081–1085.
Rhodes, D., Schwabe, J W.R., Chapman, L. and Fairall, L. (1996) Towards understanding of protein-DNA recognition, Phil. Trans. R. Soc. Lond. B 351, 501
Harrison, S.C. and Aggarwal, A.K. (1990) DNA recognition by proteins with the helixturn-helix motif, Annu. Rev. Biochem 59, 933–969.
Simoncsits, A., Chen, J., Percipalle P. Wang, S. Törö, I. and Pongor, S. (1997) Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators, J. Mol. Biol 267, 118–131.
Percipalle, P., Simoncsits, A., Zakhariev, S., Guarnaccia, C., Sanchez, R. and Pongor, S. (1995) Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding, EMBO J 14, 3200–3205.
Chen, J., Pongor, S. and Simoncsits, A. (1997) Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs, Nucleic Acids Res 25, 2047–2054.
Wharton, R.P. and Ptashne, M. (1985) Changing the binding specificity of a repressor by redesigning an a-helix, Nature, 316, 601–605.
Wharton, R. P. and Ptashne, M. (1987) A new specificity mutant of 434 repressor that defines an amino acid-base pair contact, Nature, 326, 888–891.
Huang, L., Sera, T. and Schultz, P.G. (1994) A permutational approach toward protein-DNA recognition, Proc. Natl. Acad Sci. USA, 91, 3969–3973.
Aggarwal, A.K., Rodger, D.W., Drottar, M., Ptashne, M. and Harrison, S.C. (1988) Recognition of DNA operator by the repressor of phage 434: a view at high resolution, Science, 242, 899–907.
Choo, Y. and Klug, A. (1994) Toward a code for interaction of zinc fingers with DNA: selection of randomized fingers displayed on phage, Proc. Natl. Acad. Sci. USA, 91 11163–11167.
Choo, Y. and Klug, A. (1994) Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions, Proc. Natl. Acad. Sci. USA, 91,11168–11172.
Seeman, N.C., Rosenberg, M.J. and Rich, A. (1976) Sequence-specific recognition of double helical nucleic acids by proteins, Proc. Natl. Acad. Sci. USA,73 804–808.
Pabo, C.O. and Sauer., R.T. (1992) Transcription factors: structural families and principles of DNA recognition, Annu. Rev. Biochem 61 1053–1095.
Suzuki, M. (1994) A framework for the DNA-protein recognition code of the probe helix in transcription factors: the chemical and stereochemical rules, Structure, 2 317–326.
Mandel-Gutfreund, Y., Schueler, O. and Margalit, H. (1995) Comprehensive analysis of hydrogen bonds in regulatory protein DNA-complexes: in search of common principles, J. Mol. Biol 253 370–382.
Shimon, L.J. and Harrison, S.C. (1993) The phage 434 OR2/R1–69 complex at 2.5 A resolution, T. Mol. Biol 232 826–838.
Rodgers, D.W. and Harrison, S.C. (1993) The complex between phage 434 repressor DNA-binding domain and operator site OR3: structural differences between consensus and non-consensus half-sites, Structure 1 227–240.
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Gill, S.C. and von Hippel, P.H. (1989) Calculation of protein extinction coefficients from amino acid sequence data, Anal. Biochem 182 319–326.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Springer Science+Business Media Dordrecht
About this chapter
Cite this chapter
Simoncsits, A., Tjörnhammar, ML., Wang, S., Pongor, S. (1999). Single-Chain 434 Repressors with Altered DNA-Binding Specificities. In: Bradbury, E.M., Pongor, S. (eds) Structural Biology and Functional Genomics. NATO Science Series, vol 71. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-4631-9_8
Download citation
DOI: https://doi.org/10.1007/978-94-011-4631-9_8
Publisher Name: Springer, Dordrecht
Print ISBN: 978-0-7923-5782-7
Online ISBN: 978-94-011-4631-9
eBook Packages: Springer Book Archive