Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Nov 25;17(22):9447–9468. doi: 10.1093/nar/17.22.9447

Influence of the sequence-dependent flexure of DNA on transcription in E. coli.

C M Collis 1, P L Molloy 1, G W Both 1, H R Drew 1
PMCID: PMC335145  PMID: 2685760

Abstract

In order to study the effects of DNA structure on cellular processes such as transcription, we have made a series of plasmids that locate several different kinds of DNA structure (stiff, flexible or curved) near the sites of cleavage by commonly-used restriction enzymes. One can use these plasmids to place any DNA region of interest (e.g., promoter, operator or enhancer) close to certain kinds of DNA structure that may influence its ability to work in a living cell. In the present example, we have placed a promoter from T7 virus next to the special DNA structures; the T7 promoter is then linked to a gene for a marker protein (chloramphenicol acetyl transferase). When plasmids bearing the T7 promoter are grown in cells of E. coli that contain T7 RNA polymerase, the special DNA structures seem to have little or no influence over the activity of the T7 promoter, contrary to our expectations. Yet when the same plasmids are grown in cells of E. coli that do not contain T7 RNA polymerase, some of the DNA structures show a surprising promoter activity of their own. In particular, the favourable flexibility or curvature of DNA, in the close vicinity of potential -35 and -10 promoter regions, seems to be a significant factor in determining where E. coli RNA polymerase starts RNA chains. We show directly, in one example, that loss of curvature between -35 and -10 regions is associated with a nearly-complete loss of promoter activity. These results, and others of their kind, show that the structural and/or vibrational properties of DNA play a much more important role in determining E. coli promoter activity than has previously been supposed.

Full text

PDF
9447

Images in this article

9459Image
on p.9459

Selected References

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

  1. Amouyal M., Buc H. Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli. J Mol Biol. 1987 Jun 20;195(4):795–808. doi: 10.1016/0022-2836(87)90485-2. [DOI] [PubMed] [Google Scholar]
  2. Aoyama T., Takanami M. Supercoiling response of E. coli promoters with different spacer lengths. Biochim Biophys Acta. 1988 Mar 31;949(3):311–317. doi: 10.1016/0167-4781(88)90157-1. [DOI] [PubMed] [Google Scholar]
  3. Auble D. T., Allen T. L., deHaseth P. L. Promoter recognition by Escherichia coli RNA polymerase. Effects of substitutions in the spacer DNA separating the -10 and -35 regions. J Biol Chem. 1986 Aug 25;261(24):11202–11206. [PubMed] [Google Scholar]
  4. Bauer B. F., Kar E. G., Elford R. M., Holmes W. M. Sequence determinants for promoter strength in the leuV operon of Escherichia coli. Gene. 1988;63(1):123–134. doi: 10.1016/0378-1119(88)90551-3. [DOI] [PubMed] [Google Scholar]
  5. Bossi L., Smith D. M. Conformational change in the DNA associated with an unusual promoter mutation in a tRNA operon of Salmonella. Cell. 1984 Dec;39(3 Pt 2):643–652. doi: 10.1016/0092-8674(84)90471-9. [DOI] [PubMed] [Google Scholar]
  6. Broyles S. S., Pettijohn D. E. Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered DNA helical pitch. J Mol Biol. 1986 Jan 5;187(1):47–60. doi: 10.1016/0022-2836(86)90405-5. [DOI] [PubMed] [Google Scholar]
  7. Burkhoff A. M., Tullius T. D. The unusual conformation adopted by the adenine tracts in kinetoplast DNA. Cell. 1987 Mar 27;48(6):935–943. doi: 10.1016/0092-8674(87)90702-1. [DOI] [PubMed] [Google Scholar]
  8. Calladine C. R., Drew H. R., McCall M. J. The intrinsic curvature of DNA in solution. J Mol Biol. 1988 May 5;201(1):127–137. doi: 10.1016/0022-2836(88)90444-5. [DOI] [PubMed] [Google Scholar]
  9. Calladine C. R., Drew H. R. Principles of sequence-dependent flexure of DNA. J Mol Biol. 1986 Dec 20;192(4):907–918. doi: 10.1016/0022-2836(86)90036-7. [DOI] [PubMed] [Google Scholar]
  10. Chapman K. A., Burgess R. R. Construction of bacteriophage T7 late promoters with point mutations and characterization by in vitro transcription properties. Nucleic Acids Res. 1987 Jul 10;15(13):5413–5432. doi: 10.1093/nar/15.13.5413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen W., Tabor S., Struhl K. Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. Cell. 1987 Sep 25;50(7):1047–1055. doi: 10.1016/0092-8674(87)90171-1. [DOI] [PubMed] [Google Scholar]
  12. Cruse W. B., Salisbury S. A., Brown T., Cosstick R., Eckstein F., Kennard O. Chiral phosphorothioate analogues of B-DNA. The crystal structure of Rp-d[Gp(S)CpGp(S)CpGp(S)C]. J Mol Biol. 1986 Dec 20;192(4):891–905. doi: 10.1016/0022-2836(86)90035-5. [DOI] [PubMed] [Google Scholar]
  13. Deuschle U., Kammerer W., Gentz R., Bujard H. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986 Nov;5(11):2987–2994. doi: 10.1002/j.1460-2075.1986.tb04596.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dickerson R. E., Drew H. R. Structure of a B-DNA dodecamer. II. Influence of base sequence on helix structure. J Mol Biol. 1981 Jul 15;149(4):761–786. doi: 10.1016/0022-2836(81)90357-0. [DOI] [PubMed] [Google Scholar]
  15. Drew H. R., Calladine C. R. Sequence-specific positioning of core histones on an 860 base-pair DNA. Experiment and theory. J Mol Biol. 1987 May 5;195(1):143–173. doi: 10.1016/0022-2836(87)90333-0. [DOI] [PubMed] [Google Scholar]
  16. Drew H. R., Dickerson R. E. Structure of a B-DNA dodecamer. III. Geometry of hydration. J Mol Biol. 1981 Sep 25;151(3):535–556. doi: 10.1016/0022-2836(81)90009-7. [DOI] [PubMed] [Google Scholar]
  17. Drew H. R., Travers A. A. DNA bending and its relation to nucleosome positioning. J Mol Biol. 1985 Dec 20;186(4):773–790. doi: 10.1016/0022-2836(85)90396-1. [DOI] [PubMed] [Google Scholar]
  18. Drew H. R., Travers A. A. DNA structural variations in the E. coli tyrT promoter. Cell. 1984 Jun;37(2):491–502. doi: 10.1016/0092-8674(84)90379-9. [DOI] [PubMed] [Google Scholar]
  19. Drew H. R., Travers A. A. Structural junctions in DNA: the influence of flanking sequence on nuclease digestion specificities. Nucleic Acids Res. 1985 Jun 25;13(12):4445–4467. doi: 10.1093/nar/13.12.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Drew H. R., Weeks J. R., Travers A. A. Negative supercoiling induces spontaneous unwinding of a bacterial promoter. EMBO J. 1985 Apr;4(4):1025–1032. doi: 10.1002/j.1460-2075.1985.tb03734.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gourse R. L., de Boer H. A., Nomura M. DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination. Cell. 1986 Jan 17;44(1):197–205. doi: 10.1016/0092-8674(86)90498-8. [DOI] [PubMed] [Google Scholar]
  24. Greaves D. R., Patient R. K., Lilley D. M. Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene. J Mol Biol. 1985 Oct 5;185(3):461–478. doi: 10.1016/0022-2836(85)90064-6. [DOI] [PubMed] [Google Scholar]
  25. Griffith J. D. Visualization of prokaryotic DNA in a regularly condensed chromatin-like fiber. Proc Natl Acad Sci U S A. 1976 Feb;73(2):563–567. doi: 10.1073/pnas.73.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hagerman P. J. Sequence dependence of the curvature of DNA: a test of the phasing hypothesis. Biochemistry. 1985 Dec 3;24(25):7033–7037. doi: 10.1021/bi00346a001. [DOI] [PubMed] [Google Scholar]
  27. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Koo H. S., Crothers D. M. Calibration of DNA curvature and a unified description of sequence-directed bending. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1763–1767. doi: 10.1073/pnas.85.6.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Lamond A. I., Travers A. A. Requirement for an upstream element for optimal transcription of a bacterial tRNA gene. Nature. 1983 Sep 15;305(5931):248–250. doi: 10.1038/305248a0. [DOI] [PubMed] [Google Scholar]
  33. Lomonossoff G. P., Butler P. J., Klug A. Sequence-dependent variation in the conformation of DNA. J Mol Biol. 1981 Jul 15;149(4):745–760. doi: 10.1016/0022-2836(81)90356-9. [DOI] [PubMed] [Google Scholar]
  34. Lozinski T., Markiewicz W. T., Wyrzykiewicz T. K., Wierzchowski K. L. Effect of the sequence-dependent structure of the 17 bp AT spacer on the strength of consensuslike E.coli promoters in vivo. Nucleic Acids Res. 1989 May 25;17(10):3855–3863. doi: 10.1093/nar/17.10.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marini J. C., Levene S. D., Crothers D. M., Englund P. T. Bent helical structure in kinetoplast DNA. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7664–7668. doi: 10.1073/pnas.79.24.7664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McAllister C. F., Achberger E. C. Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis. J Biol Chem. 1988 Aug 25;263(24):11743–11749. [PubMed] [Google Scholar]
  37. McAllister C. F., Achberger E. C. Rotational orientation of upstream curved DNA affects promoter function in Bacillus subtilis. J Biol Chem. 1989 Jun 25;264(18):10451–10456. [PubMed] [Google Scholar]
  38. McAllister W. T., Morris C., Rosenberg A. H., Studier F. W. Utilization of bacteriophage T7 late promoters in recombinant plasmids during infection. J Mol Biol. 1981 Dec 15;153(3):527–544. doi: 10.1016/0022-2836(81)90406-x. [DOI] [PubMed] [Google Scholar]
  39. McCall M., Brown T., Kennard O. The crystal structure of d(G-G-G-G-C-C-C-C). A model for poly(dG).poly(dC). J Mol Biol. 1985 Jun 5;183(3):385–396. doi: 10.1016/0022-2836(85)90009-9. [DOI] [PubMed] [Google Scholar]
  40. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nelson H. C., Finch J. T., Luisi B. F., Klug A. The structure of an oligo(dA).oligo(dT) tract and its biological implications. Nature. 1987 Nov 19;330(6145):221–226. doi: 10.1038/330221a0. [DOI] [PubMed] [Google Scholar]
  42. Rhodes D. Nucleosome cores reconstituted from poly (dA-dT) and the octamer of histones. Nucleic Acids Res. 1979;6(5):1805–1816. doi: 10.1093/nar/6.5.1805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rouvière-Yaniv J., Yaniv M., Germond J. E. E. coli DNA binding protein HU forms nucleosomelike structure with circular double-stranded DNA. Cell. 1979 Jun;17(2):265–274. doi: 10.1016/0092-8674(79)90152-1. [DOI] [PubMed] [Google Scholar]
  44. Russell D. W., Smith M., Cox D., Williamson V. M., Young E. T. DNA sequences of two yeast promoter-up mutants. Nature. 1983 Aug 18;304(5927):652–654. doi: 10.1038/304652a0. [DOI] [PubMed] [Google Scholar]
  45. Satchwell S. C., Drew H. R., Travers A. A. Sequence periodicities in chicken nucleosome core DNA. J Mol Biol. 1986 Oct 20;191(4):659–675. doi: 10.1016/0022-2836(86)90452-3. [DOI] [PubMed] [Google Scholar]
  46. Simpson R. T., Künzler P. Cromatin and core particles formed from the inner histones and synthetic polydeoxyribonucleotides of defined sequence. Nucleic Acids Res. 1979 Apr;6(4):1387–1415. doi: 10.1093/nar/6.4.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Struhl K. Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8419–8423. doi: 10.1073/pnas.82.24.8419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  49. Summers D. K., Sherratt D. J. Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site. EMBO J. 1988 Mar;7(3):851–858. doi: 10.1002/j.1460-2075.1988.tb02884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Summers W. C. A simple method for extraction of RNA from E. coli utilizing diethyl pyrocarbonate. Anal Biochem. 1970 Feb;33(2):459–463. doi: 10.1016/0003-2697(70)90316-7. [DOI] [PubMed] [Google Scholar]
  51. Wahle E., Kornberg A. The partition locus of plasmid pSC101 is a specific binding site for DNA gyrase. EMBO J. 1988 Jun;7(6):1889–1895. doi: 10.1002/j.1460-2075.1988.tb03022.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang A. H., Fujii S., van Boom J. H., Rich A. Molecular structure of the octamer d(G-G-C-C-G-G-C-C): modified A-DNA. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3968–3972. doi: 10.1073/pnas.79.13.3968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zahn K., Blattner F. R. Direct evidence for DNA bending at the lambda replication origin. Science. 1987 Apr 24;236(4800):416–422. doi: 10.1126/science.2951850. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES