Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(4):1253–1261. doi: 10.1128/jb.179.4.1253-1261.1997

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

A L Donner 1, P A Carlson 1, G B Koudelka 1
PMCID: PMC178823  PMID: 9023209

Abstract

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Battista J. R., Ohta T., Nohmi T., Sun W., Walker G. C. Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7190–7194. doi: 10.1073/pnas.87.18.7190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bell A. C., Koudelka G. B. Operator sequence context influences amino acid-base-pair interactions in 434 repressor-operator complexes. J Mol Biol. 1993 Dec 5;234(3):542–553. doi: 10.1006/jmbi.1993.1610. [DOI] [PubMed] [Google Scholar]
  3. Benson N., Adams C., Youderian P. Genetic selection for mutations that impair the co-operative binding of lambda repressor. Mol Microbiol. 1994 Feb;11(3):567–579. doi: 10.1111/j.1365-2958.1994.tb00337.x. [DOI] [PubMed] [Google Scholar]
  4. Carlson P. A., Koudelka G. B. Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor. J Bacteriol. 1994 Nov;176(22):6907–6914. doi: 10.1128/jb.176.22.6907-6914.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coulondre C., Miller J. H. Genetic studies of the lac repressor. III. Additional correlation of mutational sites with specific amino acid residues. J Mol Biol. 1977 Dec 15;117(3):525–567. doi: 10.1016/0022-2836(77)90056-0. [DOI] [PubMed] [Google Scholar]
  6. Cuenoud B., Schepartz A. Altered specificity of DNA-binding proteins with transition metal dimerization domains. Science. 1993 Jan 22;259(5094):510–513. doi: 10.1126/science.8424173. [DOI] [PubMed] [Google Scholar]
  7. Dahlman-Wright K., Wright A., Gustafsson J. A., Carlstedt-Duke J. Interaction of the glucocorticoid receptor DNA-binding domain with DNA as a dimer is mediated by a short segment of five amino acids. J Biol Chem. 1991 Feb 15;266(5):3107–3112. [PubMed] [Google Scholar]
  8. De Anda J., Poteete A. R., Sauer R. T. P22 c2 repressor. Domain structure and function. J Biol Chem. 1983 Sep 10;258(17):10536–10542. [PubMed] [Google Scholar]
  9. Finger L. R., Richardson J. P. Stabilization of the hexameric form of Escherichia coli protein rho under ATP hydrolysis conditions. J Mol Biol. 1982 Mar 25;156(1):203–219. doi: 10.1016/0022-2836(82)90467-3. [DOI] [PubMed] [Google Scholar]
  10. Freedman L. P., Luisi B. F. On the mechanism of DNA binding by nuclear hormone receptors: a structural and functional perspective. J Cell Biochem. 1993 Feb;51(2):140–150. doi: 10.1002/jcb.240510205. [DOI] [PubMed] [Google Scholar]
  11. Hai T., Curran T. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3720–3724. doi: 10.1073/pnas.88.9.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Johnson A. D., Meyer B. J., Ptashne M. Interactions between DNA-bound repressors govern regulation by the lambda phage repressor. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5061–5065. doi: 10.1073/pnas.76.10.5061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson A. D., Pabo C. O., Sauer R. T. Bacteriophage lambda repressor and cro protein: interactions with operator DNA. Methods Enzymol. 1980;65(1):839–856. doi: 10.1016/s0076-6879(80)65078-2. [DOI] [PubMed] [Google Scholar]
  15. Kim J., Tzamarias D., Ellenberger T., Harrison S. C., Struhl K. Adaptability at the protein-DNA interface is an important aspect of sequence recognition by bZIP proteins. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4513–4517. doi: 10.1073/pnas.90.10.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koudelka G. B., Carlson P. DNA twisting and the effects of non-contacted bases on affinity of 434 operator for 434 repressor. Nature. 1992 Jan 2;355(6355):89–91. doi: 10.1038/355089a0. [DOI] [PubMed] [Google Scholar]
  17. Koudelka G. B., Harbury P., Harrison S. C., Ptashne M. DNA twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4633–4637. doi: 10.1073/pnas.85.13.4633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koudelka G. B., Harrison S. C., Ptashne M. Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro. 1987 Apr 30-May 6Nature. 326(6116):886–888. doi: 10.1038/326886a0. [DOI] [PubMed] [Google Scholar]
  19. Little J. W. Autodigestion of lexA and phage lambda repressors. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1375–1379. doi: 10.1073/pnas.81.5.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pabo C. O., Sauer R. T., Sturtevant J. M., Ptashne M. The lambda repressor contains two domains. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1608–1612. doi: 10.1073/pnas.76.4.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peat T. S., Frank E. G., McDonald J. P., Levine A. S., Woodgate R., Hendrickson W. A. Structure of the UmuD' protein and its regulation in response to DNA damage. Nature. 1996 Apr 25;380(6576):727–730. doi: 10.1038/380727a0. [DOI] [PubMed] [Google Scholar]
  22. Poteete A. R., Ptashne M., Ballivet M., Eisen H. Operator sequences of bacteriophages P22 and 21. J Mol Biol. 1980 Feb 15;137(1):81–91. doi: 10.1016/0022-2836(80)90158-8. [DOI] [PubMed] [Google Scholar]
  23. Rastinejad F., Perlmann T., Evans R. M., Sigler P. B. Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature. 1995 May 18;375(6528):203–211. doi: 10.1038/375203a0. [DOI] [PubMed] [Google Scholar]
  24. Sauer R. T., Pabo C. O., Meyer B. J., Ptashne M., Backman K. C. Regulatory functions of the lambda repressor reside in the amino-terminal domain. Nature. 1979 May 31;279(5712):396–400. doi: 10.1038/279396a0. [DOI] [PubMed] [Google Scholar]
  25. Sauer R. T., Ross M. J., Ptashne M. Cleavage of the lambda and P22 repressors by recA protein. J Biol Chem. 1982 Apr 25;257(8):4458–4462. [PubMed] [Google Scholar]
  26. Schwabe J. W., Chapman L., Finch J. T., Rhodes D. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell. 1993 Nov 5;75(3):567–578. doi: 10.1016/0092-8674(93)90390-c. [DOI] [PubMed] [Google Scholar]
  27. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Valenzuela D., Ptashne M. P22 repressor mutants deficient in co-operative binding and DNA loop formation. EMBO J. 1989 Dec 20;8(13):4345–4350. doi: 10.1002/j.1460-2075.1989.tb08621.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wharton R. P., Brown E. L., Ptashne M. Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor. Cell. 1984 Sep;38(2):361–369. doi: 10.1016/0092-8674(84)90491-4. [DOI] [PubMed] [Google Scholar]
  30. Whipple F. W., Kuldell N. H., Cheatham L. A., Hochschild A. Specificity determinants for the interaction of lambda repressor and P22 repressor dimers. Genes Dev. 1994 May 15;8(10):1212–1223. doi: 10.1101/gad.8.10.1212. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES