Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Nov;175(21):6932–6938. doi: 10.1128/jb.175.21.6932-6938.1993

Characterization of the Tn5 transposase and inhibitor proteins: a model for the inhibition of transposition.

N B de la Cruz 1, M D Weinreich 1, T W Wiegand 1, M P Krebs 1, W S Reznikoff 1
PMCID: PMC206819  PMID: 8226636

Abstract

Tn5 is a composite transposon consisting of two IS50 sequences in inverted orientation with respect to a unique, central region encoding several antibiotic resistances. The IS50R element encodes two proteins in the same reading frame which regulate the transposition reaction: the transposase (Tnp), which is required for transposition, and an inhibitor of transposition (Inh). The inhibitor is a naturally occurring deletion variant of Tnp which lacks the N-terminal 55 amino acids. In this report, we present the purification of both the Tnp and Inh proteins and an analysis of their DNA binding properties. Purified Tnp, but not Inh, was found to bind specifically to the outside end of Tn5. Inh, however, stimulated the binding activity of Tnp to outside-end DNA and was shown to be present with Tnp in these bound complexes. Inh was also found to exist as a dimer in solution. These results indicate that the N-terminal 55 amino acids of Tnp are required for sequence-specific binding. They also suggest that Inh inhibits transposition by forming mixed oligomers with Tnp which still bind to the ends of the transposon but are defective for later stages of the transposition reaction.

Full text

PDF
6932

Images in this article

6934Image
on p.6934
6935Image
on p.6935
6936Image
on p.6936

Selected References

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

  1. Baker T. A., Mizuuchi K. DNA-promoted assembly of the active tetramer of the Mu transposase. Genes Dev. 1992 Nov;6(11):2221–2232. doi: 10.1101/gad.6.11.2221. [DOI] [PubMed] [Google Scholar]
  2. Biek D., Roth J. R. Regulation of Tn5 transposition in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1980 Oct;77(10):6047–6051. doi: 10.1073/pnas.77.10.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. DeLong A., Syvanen M. Trans-acting transposase mutant from Tn5. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6072–6076. doi: 10.1073/pnas.88.14.6072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grodberg J., Dunn J. J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245–1253. doi: 10.1128/jb.170.3.1245-1253.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  7. Isberg R. R., Lazaar A. L., Syvanen M. Regulation of Tn5 by the right-repeat proteins: control at the level of the transposition reaction? Cell. 1982 Oct;30(3):883–892. doi: 10.1016/0092-8674(82)90293-8. [DOI] [PubMed] [Google Scholar]
  8. Johnson R. C., Reznikoff W. S. DNA sequences at the ends of transposon Tn5 required for transposition. Nature. 1983 Jul 21;304(5923):280–282. doi: 10.1038/304280a0. [DOI] [PubMed] [Google Scholar]
  9. Johnson R. C., Reznikoff W. S. Role of the IS50 R proteins in the promotion and control of Tn5 transposition. J Mol Biol. 1984 Aug 25;177(4):645–661. doi: 10.1016/0022-2836(84)90042-1. [DOI] [PubMed] [Google Scholar]
  10. Johnson R. C., Yin J. C., Reznikoff W. S. Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat. Cell. 1982 Oct;30(3):873–882. doi: 10.1016/0092-8674(82)90292-6. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Rothstein S. J., Reznikoff W. S. The functional differences in the inverted repeats of Tn5 are caused by a single base pair nonhomology. Cell. 1981 Jan;23(1):191–199. doi: 10.1016/0092-8674(81)90284-1. [DOI] [PubMed] [Google Scholar]
  15. Sasakawa C., Carle G. F., Berg D. E. Sequences essential for transposition at the termini of IS50. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7293–7297. doi: 10.1073/pnas.80.23.7293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schulz V. P., Reznikoff W. S. Translation initiation of IS50R read-through transcripts. J Mol Biol. 1991 Sep 5;221(1):65–80. doi: 10.1016/0022-2836(91)80205-9. [DOI] [PubMed] [Google Scholar]
  17. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Yin J. C., Reznikoff W. S. p2 and inhibition of Tn5 transposition. J Bacteriol. 1988 Jul;170(7):3008–3015. doi: 10.1128/jb.170.7.3008-3015.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES