Skip to main content
SpringerLink
Log in

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

  • Published:
Science in China Series C: Life Sciences Aims and scope Submit manuscript

Abstract

Single-chain repressor RRTres is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1–69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the −1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic,in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTres The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1 × 10−12 mol/L-1 × 1011 mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1 × 10−9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recognition properties previously determined for the RTREs domain. These operators containing the consensus sequenceGTAAGAAARNTTACN orGGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA interactions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

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

  1. Aggarwal, A. K., Rodgers, D. W., Drottar, M. et al., Recognition of a DNA operator by the repressor of phage 434: A view at high resolution, Science, 1988, 242: 899–907.

    Article  PubMed  CAS  Google Scholar 

  2. Anderson, J. E., Ptashne, M., Harrison, S. C., Structure of the repressor-operator complex of bacteriophage 434, Nature, 1987, 326: 846–852.

    Article  PubMed  CAS  Google Scholar 

  3. Bushman, F. D., The Bacteriophage 434 right operator roles of OR1, OR2 and OR3, J. Mol. Biol., 1993, 230: 28–40.

    Article  PubMed  CAS  Google Scholar 

  4. Bell, A. C., Koudelka, G. B., How 434 repressor discriminates between OR1 and OR3, J. Biological Chemistry, 1995, 270: 1205–1212.

    Article  CAS  Google Scholar 

  5. Bell, A. C., Koudelka, G. B., Operator sequence context influences amino acid-base-pair interaction in 434 repressor-operator complexes, J. Mol. Biol., 1993, 234: 542–553.

    Article  PubMed  CAS  Google Scholar 

  6. Wharton, R. P., Ptashne, M., A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact, Nature, 1987, 326: 888–891.

    Article  PubMed  CAS  Google Scholar 

  7. Wharton, R. P., Brown, E. L., Ptashne, M., Substituting an α-helix switches the sequence-specific DNA interaction of a repressor, Cell., 1984, 38: 361–369.

    Article  PubMed  CAS  Google Scholar 

  8. Hollis, M., Valenzuela, D., Pioli, D. et al., A repressor heterodimer binds to a chimeric operator, Proc. Natl. Acad. Sci. USA, 1988, 85: 5834–5838.

    Article  PubMed  CAS  Google Scholar 

  9. Huang, L. -X., Sera, T., Schultz, P. G., A permutational approach toward protein-DNA recognition, Proc. Natl. Acad. Sci. USA, 1994, 91: 3969–3973.

    Article  PubMed  CAS  Google Scholar 

  10. Percipalle, P., Simoncsits, A., Zakhariev, S. et al., Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding, EMBO J., 1995, 14: 3200–3205.

    PubMed  CAS  Google Scholar 

  11. Simoncsits, A., Chen, J. -Q., Percipalle, P. et al., Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators, J. Mol. Biol., 1997, 267: 118–131.

    Article  PubMed  CAS  Google Scholar 

  12. Gates, C. M., Stemmer, W. P. C., Kaptein, R. et al., Affinity selective isolation of ligands from peptide libraries through display on a lac repressor “headpiece dimmer”, J. Mol. Biol., 1996, 255: 373–386.

    Article  PubMed  CAS  Google Scholar 

  13. Kim, J. -S., Pabo, C. O., Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants, Proc. Natl. Acad. Sci. USA, 1998, 95: 2812–2817.

    Article  PubMed  CAS  Google Scholar 

  14. Wu, H., Yang, W. -P., BarbasIII, C. F., Building zinc fingers by selection: toward a therapeutic application, Proc. Natl. Acad. Sci. USA, 1995, 92: 344–348.

    Article  PubMed  CAS  Google Scholar 

  15. Wang, B. S., Pabo, C. O., Dimerization of zinc fingers mediated by peptides evolved in vitro from random sequences, Proc. Natl. Acad. Sci. USA, 1999, 96: 9568–9573.

    Article  PubMed  CAS  Google Scholar 

  16. Choo, Y., Sánchez-García, I., Klug, A., In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence, Nature, 1994, 372: 642–645.

    Article  PubMed  CAS  Google Scholar 

  17. Wolfe, S. A., Greisman, H. A., Ramm, E. I. et al., Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code, J. Mol. Biol., 1999, 285: 1917–1934.

    Article  PubMed  CAS  Google Scholar 

  18. Chen, J. -Q., Pongor, S., Simoncsits, A., Recognition of DNAby single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs, Nucleic Acids Research, 1997, 25: 2047–2054.

    Article  PubMed  CAS  Google Scholar 

  19. Simoncsits, A., Tjörnhammar, M. -L., Wang, S. -L. et al., Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites, Nucleic Acids Research, 1999, 27: 3474–3480.

    Article  PubMed  CAS  Google Scholar 

  20. Zhou, Y. -H., Busby, S., Ebright, R. H., Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers, Cell, 1993, 73: 375–379.

    Article  PubMed  CAS  Google Scholar 

  21. Studier, F. W., Rosenberg, A. H., Dunn, J. J. et al., Use of T7 RNA polymerase to direct expression of cloned genes, Methods Enzymol., 1990, 185: 60–89.

    Article  PubMed  CAS  Google Scholar 

  22. Simoncsits, A., Bristulf, J., Tjörnhammar, M. -L. et al., Deletion mutants of human interleukin 1 with significantly reduced agonist properties: search for agonist/ antagonist switch in ligands to the interleukin 1 receptors, Cytokine, 1994, 6: 206–214.

    Article  PubMed  CAS  Google Scholar 

  23. Sanger, F., Nicklen, S., Coulson, A. R., DNA sequencing with chain terminating inhibitors, Proc. Natl. Acad. Sci. USA, 1977, 74: 5463–5467.

    Article  PubMed  CAS  Google Scholar 

  24. Carey, J., Gel retardation, Methods Enzymol., 1991, 208: 103–117.

    Article  PubMed  CAS  Google Scholar 

  25. Mandel-Gutfreund, Y., Schueler, O., Margalit, H., Comprehensive analysis of hydrogen bonds in regulatory protein DNA-complexes: in search of common principles, J. Mol. Biol., 1995, 253: 370–382.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Botany, Chinese Academy of Sciences, 100093, Beijing, China

    Tiebing Liang, Kehui Tan, Kang Chong & Zhiqing Zhu

  2. ICGEB, 34012, Trieste, Italy

    Sándor Pongor & András Simoncsits

Authors
  1. Tiebing Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kehui Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kang Chong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhiqing Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sándor Pongor
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. András Simoncsits
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to András Simoncsits.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liang, T., Tan, K., Chong, K. et al. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor. Sci. China Ser. C.-Life Sci. 44, 274–286 (2001). https://doi.org/10.1007/BF02879334

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02879334

Keywords

Search

Navigation