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. 1988 Jan;7(1):269–275. doi: 10.1002/j.1460-2075.1988.tb02809.x

Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA.

S Hurstel 1, M Granger-Schnarr 1, M Schnarr 1
PMCID: PMC454268  PMID: 3282882

Abstract

Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA. These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation. This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface. The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers. Each half-operator contains seven interfering phosphates. These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target. On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis. We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein. A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain. We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay.

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Selected References

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

  1. Anderson J. E., Ptashne M., Harrison S. C. Structure of the repressor-operator complex of bacteriophage 434. 1987 Apr 30-May 6Nature. 326(6116):846–852. doi: 10.1038/326846a0. [DOI] [PubMed] [Google Scholar]
  2. Bertrand-Burggraf E., Hurstel S., Daune M., Schnarr M. Promoter properties and negative regulation of the uvrA gene by the LexA repressor and its amino-terminal DNA binding domain. J Mol Biol. 1987 Jan 20;193(2):293–302. doi: 10.1016/0022-2836(87)90220-8. [DOI] [PubMed] [Google Scholar]
  3. Boelens R., Scheek R. M., van Boom J. H., Kaptein R. Complex of lac repressor headpiece with a 14 base-pair lac operator fragment studied by two-dimensional nuclear magnetic resonance. J Mol Biol. 1987 Jan 5;193(1):213–216. doi: 10.1016/0022-2836(87)90638-3. [DOI] [PubMed] [Google Scholar]
  4. Bushman F. D., Anderson J. E., Harrison S. C., Ptashne M. Ethylation interference and X-ray crystallography identify similar interactions between 434 repressor and operator. Nature. 1985 Aug 15;316(6029):651–653. doi: 10.1038/316651a0. [DOI] [PubMed] [Google Scholar]
  5. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  6. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  7. Dodd I. B., Egan J. B. Systematic method for the detection of potential lambda Cro-like DNA-binding regions in proteins. J Mol Biol. 1987 Apr 5;194(3):557–564. doi: 10.1016/0022-2836(87)90681-4. [DOI] [PubMed] [Google Scholar]
  8. Granger-Schnarr M., Schnarr M., van Sluis C. A. In vitro study of the interaction of the LexA repressor and the UvrC protein with a uvrC regulatory region. FEBS Lett. 1986 Mar 17;198(1):61–65. doi: 10.1016/0014-5793(86)81185-1. [DOI] [PubMed] [Google Scholar]
  9. Hertzberg R. P., Dervan P. B. Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses. Biochemistry. 1984 Aug 14;23(17):3934–3945. doi: 10.1021/bi00312a022. [DOI] [PubMed] [Google Scholar]
  10. Hochschild A., Douhan J., 3rd, Ptashne M. How lambda repressor and lambda Cro distinguish between OR1 and OR3. Cell. 1986 Dec 5;47(5):807–816. doi: 10.1016/0092-8674(86)90523-4. [DOI] [PubMed] [Google Scholar]
  11. Hurstel S., Granger-Schnarr M., Daune M., Schnarr M. In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain. EMBO J. 1986 Apr;5(4):793–798. doi: 10.1002/j.1460-2075.1986.tb04283.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kitagawa Y., Akaboshi E., Shinagawa H., Horii T., Ogawa H., Kato T. Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4336–4340. doi: 10.1073/pnas.82.13.4336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koo H. S., Wu H. M., Crothers D. M. DNA bending at adenine . thymine tracts. Nature. 1986 Apr 10;320(6062):501–506. doi: 10.1038/320501a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Little J. W., Hill S. A. Deletions within a hinge region of a specific DNA-binding protein. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2301–2305. doi: 10.1073/pnas.82.8.2301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
  19. Little J. W., Mount D. W., Yanisch-Perron C. R. Purified lexA protein is a repressor of the recA and lexA genes. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4199–4203. doi: 10.1073/pnas.78.7.4199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohlendorf D. H., Anderson W. F., Matthews B. W. Many gene-regulatory proteins appear to have a similar alpha-helical fold that binds DNA and evolved from a common precursor. J Mol Evol. 1983;19(2):109–114. doi: 10.1007/BF02300748. [DOI] [PubMed] [Google Scholar]
  21. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  22. Peterson K. R., Mount D. W. Differential repression of SOS genes by unstable lexA41 (tsl-1) protein causes a "split-phenotype" in Escherichia coli K-12. J Mol Biol. 1987 Jan 5;193(1):27–40. doi: 10.1016/0022-2836(87)90623-1. [DOI] [PubMed] [Google Scholar]
  23. Poteete A. R., Ptashne M. Control of transcription by the bacteriophage P22 repressor. J Mol Biol. 1982 May 5;157(1):21–48. doi: 10.1016/0022-2836(82)90511-3. [DOI] [PubMed] [Google Scholar]
  24. Ptashne M., Jeffrey A., Johnson A. D., Maurer R., Meyer B. J., Pabo C. O., Roberts T. M., Sauer R. T. How the lambda repressor and cro work. Cell. 1980 Jan;19(1):1–11. doi: 10.1016/0092-8674(80)90383-9. [DOI] [PubMed] [Google Scholar]
  25. Schnarr M., Daune M. Cooperative and salt-resistant binding of lexA protein to non-operator DNA. FEBS Lett. 1984 Jun 11;171(2):207–210. doi: 10.1016/0014-5793(84)80489-5. [DOI] [PubMed] [Google Scholar]
  26. Schnarr M., Pouyet J., Granger-Schnarr M., Daune M. Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli. Biochemistry. 1985 May 21;24(11):2812–2818. doi: 10.1021/bi00332a032. [DOI] [PubMed] [Google Scholar]
  27. Shanblatt S. H., Revzin A. The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies. J Biol Chem. 1986 Aug 15;261(23):10885–10890. [PubMed] [Google Scholar]
  28. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tullius T. D., Dombroski B. A. Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5469–5473. doi: 10.1073/pnas.83.15.5469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tullius T. D., Dombroski B. A. Iron(II) EDTA used to measure the helical twist along any DNA molecule. Science. 1985 Nov 8;230(4726):679–681. doi: 10.1126/science.2996145. [DOI] [PubMed] [Google Scholar]
  31. Van Dyke M. W., Hertzberg R. P., Dervan P. B. Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage-inhibition patterns with methidiumpropyl-EDTA.Fe(II). Proc Natl Acad Sci U S A. 1982 Sep;79(18):5470–5474. doi: 10.1073/pnas.79.18.5470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wertman K. F., Little J. W., Mount D. W. Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3801–3805. doi: 10.1073/pnas.81.12.3801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wertman K. F., Mount D. W. Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12. J Bacteriol. 1985 Jul;163(1):376–384. doi: 10.1128/jb.163.1.376-384.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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