Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Mar;178(6):1671–1679. doi: 10.1128/jb.178.6.1671-1679.1996

The organization of the outside end of transposon Tn5.

R A Jilk 1, D York 1, W S Reznikoff 1
PMCID: PMC177853  PMID: 8626296

Abstract

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.

Full Text

The Full Text of this article is available as a PDF (538.2 KB).

Selected References

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

  1. Arciszewska L. K., Craig N. L. Interaction of the Tn7-encoded transposition protein TnsB with the ends of the transposon. Nucleic Acids Res. 1991 Sep 25;19(18):5021–5029. doi: 10.1093/nar/19.18.5021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender J., Kleckner N. Genetic evidence that Tn10 transposes by a nonreplicative mechanism. Cell. 1986 Jun 20;45(6):801–815. doi: 10.1016/0092-8674(86)90555-6. [DOI] [PubMed] [Google Scholar]
  3. Derbyshire K. M., Grindley N. D. Binding of the IS903 transposase to its inverted repeat in vitro. EMBO J. 1992 Sep;11(9):3449–3455. doi: 10.1002/j.1460-2075.1992.tb05424.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Derbyshire K. M., Hwang L., Grindley N. D. Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8049–8053. doi: 10.1073/pnas.84.22.8049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dixon W. J., Hayes J. J., Levin J. R., Weidner M. F., Dombroski B. A., Tullius T. D. Hydroxyl radical footprinting. Methods Enzymol. 1991;208:380–413. doi: 10.1016/0076-6879(91)08021-9. [DOI] [PubMed] [Google Scholar]
  6. Dodson K. W., Berg D. E. Factors affecting transposition activity of IS50 and Tn5 ends. Gene. 1989;76(2):207–213. doi: 10.1016/0378-1119(89)90161-3. [DOI] [PubMed] [Google Scholar]
  7. Dodson K. W., Berg D. E. Saturation mutagenesis of the inside end of insertion sequence IS50. Gene. 1989 Dec 21;85(1):75–81. doi: 10.1016/0378-1119(89)90466-6. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Gartenberg M. R., Ampe C., Steitz T. A., Crothers D. M. Molecular characterization of the GCN4-DNA complex. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6034–6038. doi: 10.1073/pnas.87.16.6034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goryshin IYu, Kil Y. V., Reznikoff W. S. DNA length, bending, and twisting constraints on IS50 transposition. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10834–10838. doi: 10.1073/pnas.91.23.10834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haniford D., Kleckner N. Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends. EMBO J. 1994 Jul 15;13(14):3401–3411. doi: 10.1002/j.1460-2075.1994.tb06643.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Huisman O., Errada P. R., Signon L., Kleckner N. Mutational analysis of IS10's outside end. EMBO J. 1989 Jul;8(7):2101–2109. doi: 10.1002/j.1460-2075.1989.tb03619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ichikawa H., Ikeda K., Amemura J., Ohtsubo E. Two domains in the terminal inverted-repeat sequence of transposon Tn3. Gene. 1990 Jan 31;86(1):11–17. doi: 10.1016/0378-1119(90)90108-4. [DOI] [PubMed] [Google Scholar]
  15. Jilk R. A., Makris J. C., Borchardt L., Reznikoff W. S. Implications of Tn5-associated adjacent deletions. J Bacteriol. 1993 Mar;175(5):1264–1271. doi: 10.1128/jb.175.5.1264-1271.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim J., Zwieb C., Wu C., Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene. 1989 Dec 21;85(1):15–23. doi: 10.1016/0378-1119(89)90459-9. [DOI] [PubMed] [Google Scholar]
  17. Krebs M. P., Reznikoff W. S. Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression. J Mol Biol. 1986 Dec 20;192(4):781–791. doi: 10.1016/0022-2836(86)90028-8. [DOI] [PubMed] [Google Scholar]
  18. Kuo C. F., Zou A. H., Jayaram M., Getzoff E., Harshey R. DNA-protein complexes during attachment-site synapsis in Mu DNA transposition. EMBO J. 1991 Jun;10(6):1585–1591. doi: 10.1002/j.1460-2075.1991.tb07679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Makris J. C., Nordmann P. L., Reznikoff W. S. Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2224–2228. doi: 10.1073/pnas.85.7.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  21. McCLINTOCK B. Chromosome organization and genic expression. Cold Spring Harb Symp Quant Biol. 1951;16:13–47. doi: 10.1101/sqb.1951.016.01.004. [DOI] [PubMed] [Google Scholar]
  22. Mizuuchi K. Transpositional recombination: mechanistic insights from studies of mu and other elements. Annu Rev Biochem. 1992;61:1011–1051. doi: 10.1146/annurev.bi.61.070192.005051. [DOI] [PubMed] [Google Scholar]
  23. Nordmann P. L., Makris J. C., Reznikoff W. S. Inosine induced mutations. Mol Gen Genet. 1988 Sep;214(1):62–67. doi: 10.1007/BF00340180. [DOI] [PubMed] [Google Scholar]
  24. Phadnis S. H., Berg D. E. Identification of base pairs in the outside end of insertion sequence IS50 that are needed for IS50 and Tn5 transposition. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9118–9122. doi: 10.1073/pnas.84.24.9118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reznikoff W. S. The Tn5 transposon. Annu Rev Microbiol. 1993;47:945–963. doi: 10.1146/annurev.mi.47.100193.004501. [DOI] [PubMed] [Google Scholar]
  26. Seeman N. C., Rosenberg J. M., Rich A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A. 1976 Mar;73(3):804–808. doi: 10.1073/pnas.73.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shapiro J. A. Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1933–1937. doi: 10.1073/pnas.76.4.1933. [DOI] [PMC free article] [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. Surette M. G., Harkness T., Chaconas G. Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations. J Biol Chem. 1991 Feb 15;266(5):3118–3124. [PubMed] [Google Scholar]
  30. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weinreich M. D., Gasch A., Reznikoff W. S. Evidence that the cis preference of the Tn5 transposase is caused by nonproductive multimerization. Genes Dev. 1994 Oct 1;8(19):2363–2374. doi: 10.1101/gad.8.19.2363. [DOI] [PubMed] [Google Scholar]
  32. Weinreich M. D., Mahnke-Braam L., Reznikoff W. S. A functional analysis of the Tn5 transposase. Identification of domains required for DNA binding and multimerization. J Mol Biol. 1994 Aug 12;241(2):166–177. doi: 10.1006/jmbi.1994.1486. [DOI] [PubMed] [Google Scholar]
  33. Wiater L. A., Grindley N. D. Gamma delta transposase. Purification and analysis of its interaction with a transposon end. J Biol Chem. 1991 Jan 25;266(3):1841–1849. [PubMed] [Google Scholar]
  34. Wiegand T. W., Reznikoff W. S. Characterization of two hypertransposing Tn5 mutants. J Bacteriol. 1992 Feb;174(4):1229–1239. doi: 10.1128/jb.174.4.1229-1239.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wiegand T. W., Reznikoff W. S. Interaction of Tn5 transposase with the transposon termini. J Mol Biol. 1994 Jan 14;235(2):486–495. doi: 10.1006/jmbi.1994.1008. [DOI] [PubMed] [Google Scholar]
  36. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  37. Yin J. C., Krebs M. P., Reznikoff W. S. Effect of dam methylation on Tn5 transposition. J Mol Biol. 1988 Jan 5;199(1):35–45. doi: 10.1016/0022-2836(88)90377-4. [DOI] [PubMed] [Google Scholar]
  38. Zerbib D., Prentki P., Gamas P., Freund E., Galas D. J., Chandler M. Functional organization of the ends of IS1: specific binding site for an IS 1-encoded protein. Mol Microbiol. 1990 Sep;4(9):1477–1486. [PubMed] [Google Scholar]
  39. Zhou Y., Zhang X., Ebright R. H. Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6081–6085. doi: 10.1073/pnas.90.13.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zou A. H., Leung P. C., Harshey R. M. Transposase contacts with mu DNA ends. J Biol Chem. 1991 Oct 25;266(30):20476–20482. [PubMed] [Google Scholar]
  41. de la Cruz N. B., Weinreich M. D., Wiegand T. W., Krebs M. P., Reznikoff W. S. Characterization of the Tn5 transposase and inhibitor proteins: a model for the inhibition of transposition. J Bacteriol. 1993 Nov;175(21):6932–6938. doi: 10.1128/jb.175.21.6932-6938.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES