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. 1989 Apr;8(4):1247–1255. doi: 10.1002/j.1460-2075.1989.tb03498.x

RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon.

G Kuhnke 1, C Theres 1, H J Fritz 1, R Ehring 1
PMCID: PMC400941  PMID: 2663472

Abstract

The Escherichia coli galactose operon contains an unusual array of closely spaced binding sites for proteins governing the expression from the two physically overlapping gal promoters. Based on studies of two gal promoter-up mutants we have previously suggested RNA-polymerase-induced DNA bending of gal promoter DNA. Here we present new evidence confirming and extending this interpretation. It was obtained by the circular permutation assay of gel electrophoretic mobility [Wu and Crothers (1984), Nature, 308, 509-513] applied to three analogous series of circularly permuted fragments derived from wild-type and two promoter-up mutant DNAs. The same circularly permuted DNA fragments have further been used to study the binding of gal repressor to its operator sites by electrophoretic mobility shift and by DNase I footprinting techniques. The main results are: (i) complexes carrying repressor either exclusively at the upstream operator O1 or at the downstream operator O2 exhibit different electrophoretic mobilities; (ii) binding to either one of the operators results in protein-induced DNA bending by the criteria of the circular permutation mobility assay; and (iii) occupation of both gal operators by gal repressor does not prevent cAMP-CRP-independent binding of RNA polymerase to the gal promoters, as judged by DNase I protection and gel retardation assays. The latter finding imposes constraints on any attempt to model the regulation of gal expression by assumed DNA-protein and protein-protein interactions.

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Selected References

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

  1. Crothers D. M., Fried M. Transmission of long-range effects in DNA. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):263–269. doi: 10.1101/sqb.1983.047.01.031. [DOI] [PubMed] [Google Scholar]
  2. Echols H. Multiple DNA-protein interactions governing high-precision DNA transactions. Science. 1986 Sep 5;233(4768):1050–1056. doi: 10.1126/science.2943018. [DOI] [PubMed] [Google Scholar]
  3. Fritz H. J., Bicknäse H., Gleumes B., Heibach C., Rosahl S., Ehring R. Characterization of two mutations in the Escherichia coli galE gene inactivating the second galactose operator and comparative studies of repressor binding. EMBO J. 1983;2(12):2129–2135. doi: 10.1002/j.1460-2075.1983.tb01713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  5. Gartenberg M. R., Crothers D. M. DNA sequence determinants of CAP-induced bending and protein binding affinity. Nature. 1988 Jun 30;333(6176):824–829. doi: 10.1038/333824a0. [DOI] [PubMed] [Google Scholar]
  6. Gellert M., Nash H. Communication between segments of DNA during site-specific recombination. 1987 Jan 29-Feb 4Nature. 325(6103):401–404. doi: 10.1038/325401a0. [DOI] [PubMed] [Google Scholar]
  7. Haber R., Adhya S. Interaction of spatially separated protein-DNA complexes for control of gene expression: operator conversions. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9683–9687. doi: 10.1073/pnas.85.24.9683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Heumann H., Metzger W., Niehörster M. Visualization of intermediary transcription states in the complex between Escherichia coli DNA-dependent RNA polymerases and a promoter-carrying DNA fragment using the gel retardation method. Eur J Biochem. 1986 Aug 1;158(3):575–579. doi: 10.1111/j.1432-1033.1986.tb09793.x. [DOI] [PubMed] [Google Scholar]
  9. Heumann H., Ricchetti M., Werel W. DNA-dependent RNA polymerase of Escherichia coli induces bending or an increased flexibility of DNA by specific complex formation. EMBO J. 1988 Dec 20;7(13):4379–4381. doi: 10.1002/j.1460-2075.1988.tb03336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hochschild A., Ptashne M. Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix. Cell. 1986 Mar 14;44(5):681–687. doi: 10.1016/0092-8674(86)90833-0. [DOI] [PubMed] [Google Scholar]
  11. Irani M. H., Orosz L., Adhya S. A control element within a structural gene: the gal operon of Escherichia coli. Cell. 1983 Mar;32(3):783–788. doi: 10.1016/0092-8674(83)90064-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Krämer H., Niemöller M., Amouyal M., Revet B., von Wilcken-Bergmann B., Müller-Hill B. lac repressor forms loops with linear DNA carrying two suitably spaced lac operators. EMBO J. 1987 May;6(5):1481–1491. doi: 10.1002/j.1460-2075.1987.tb02390.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuhnke G., Fritz H. J., Ehring R. Unusual properties of promoter-up mutations in the Escherichia coli galactose operon and evidence suggesting RNA polymerase-induced DNA bending. EMBO J. 1987 Feb;6(2):507–513. doi: 10.1002/j.1460-2075.1987.tb04782.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu-Johnson H. N., Gartenberg M. R., Crothers D. M. The DNA binding domain and bending angle of E. coli CAP protein. Cell. 1986 Dec 26;47(6):995–1005. doi: 10.1016/0092-8674(86)90814-7. [DOI] [PubMed] [Google Scholar]
  17. Majumdar A., Adhya S. Probing the structure of gal operator-repressor complexes. Conformation change in DNA. J Biol Chem. 1987 Sep 25;262(27):13258–13262. [PubMed] [Google Scholar]
  18. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  19. Meier I., Wray L. V., Hillen W. Differential regulation of the Tn10-encoded tetracycline resistance genes tetA and tetR by the tandem tet operators O1 and O2. EMBO J. 1988 Feb;7(2):567–572. doi: 10.1002/j.1460-2075.1988.tb02846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Musso R. E., Di Lauro R., Adhya S., de Crombrugghe B. Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters. Cell. 1977 Nov;12(3):847–854. doi: 10.1016/0092-8674(77)90283-5. [DOI] [PubMed] [Google Scholar]
  21. Piette J., Kryszke M. H., Yaniv M. Specific interaction of cellular factors with the B enhancer of polyoma virus. EMBO J. 1985 Oct;4(10):2675–2685. doi: 10.1002/j.1460-2075.1985.tb03987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Porschke D., Hillen W., Takahashi M. The change of DNA structure by specific binding of the cAMP receptor protein from rotation diffusion and dichroism measurements. EMBO J. 1984 Dec 1;3(12):2873–2878. doi: 10.1002/j.1460-2075.1984.tb02223.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ptashne M. Gene regulation by proteins acting nearby and at a distance. Nature. 1986 Aug 21;322(6081):697–701. doi: 10.1038/322697a0. [DOI] [PubMed] [Google Scholar]
  24. Richmond T. J., Finch J. T., Rushton B., Rhodes D., Klug A. Structure of the nucleosome core particle at 7 A resolution. Nature. 1984 Oct 11;311(5986):532–537. doi: 10.1038/311532a0. [DOI] [PubMed] [Google Scholar]
  25. Ryder K., Silver S., DeLucia A. L., Fanning E., Tegtmeyer P. An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. Cell. 1986 Mar 14;44(5):719–725. doi: 10.1016/0092-8674(86)90838-x. [DOI] [PubMed] [Google Scholar]
  26. Salvo J. J., Grindley N. D. Helical phasing between DNA bends and the determination of bend direction. Nucleic Acids Res. 1987 Dec 10;15(23):9771–9779. doi: 10.1093/nar/15.23.9771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shanblatt S. H., Revzin A. Role of a second catabolite activator protein molecule in controlling initiation of transcription at the galactose operon of Escherichia coli. Biochemistry. 1986 Sep 23;25(19):5539–5546. doi: 10.1021/bi00367a029. [DOI] [PubMed] [Google Scholar]
  28. Shanblatt S. H., Revzin A. Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1594–1598. doi: 10.1073/pnas.80.6.1594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Spassky A., Busby S., Buc H. On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions. EMBO J. 1984 Jan;3(1):43–50. doi: 10.1002/j.1460-2075.1984.tb01759.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Straney S. B., Crothers D. M. Lac repressor is a transient gene-activating protein. Cell. 1987 Dec 4;51(5):699–707. doi: 10.1016/0092-8674(87)90093-6. [DOI] [PubMed] [Google Scholar]
  31. Taniguchi T., de Crombrugghe B. Interactions of RNA polymerase and the cyclic AMP receptor protein on DNA of the E. coli galactose operon. Nucleic Acids Res. 1983 Aug 11;11(15):5165–5180. doi: 10.1093/nar/11.15.5165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Travers A., Klug A. Nucleoprotein complexes. DNA wrapping and writhing. 1987 May 28-Jun 3Nature. 327(6120):280–281. doi: 10.1038/327280a0. [DOI] [PubMed] [Google Scholar]
  33. Warwicker J., Engelman B. P., Steitz T. A. Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent. Proteins. 1987;2(4):283–289. doi: 10.1002/prot.340020404. [DOI] [PubMed] [Google Scholar]
  34. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  35. Zinkel S. S., Crothers D. M. DNA bend direction by phase sensitive detection. Nature. 1987 Jul 9;328(6126):178–181. doi: 10.1038/328178a0. [DOI] [PubMed] [Google Scholar]

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