Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jul 15;88(14):6072–6076. doi: 10.1073/pnas.88.14.6072

Trans-acting transposase mutant from Tn5.

A DeLong 1, M Syvanen 1
PMCID: PMC52024  PMID: 1648727

Abstract

Transposition of Tn5 and of its component insertion sequence IS50R is regulated through the action of two proteins it encodes: a cis-acting transposase, Tnp, and a trans-acting inhibitor of transposition, Inh. The mechanism of the cis-acting Tnp and the relevance of inhibition to cis action have been addressed in the current study. A specific colony morphology assay for transposition of Tn5 was shown to be sensitive to Inh produced in trans and was used to screen for mutants in Inh and/or Tnp with altered regulation. A dominant mutant in IS50R that promotes transposition in trans was isolated and characterized. The mutant (449F) carries a Leu----Phe mutation at position 449 in Tnp. This mutation reduces the frequency of Tn5 or IS50R transposition in cis but allows Tnp-449F to act as efficiently in trans as it does in cis. Tnp-449F is sensitive to inhibition and, furthermore, Inh-449F is a competent inhibitor in trans. These results show that Tnp-449F is a trans-acting transposase, unlike wild-type Tnp, which is cis-acting.

Full text

PDF

Images in this article

6074Image
on p.6074
6074Image
on p.6074
6074Image
on p.6074
6074Image
on p.6074
6074Image
on p.6074

Selected References

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

  1. Auerswald E. A., Ludwig G., Schaller H. Structural analysis of Tn5. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):107–113. doi: 10.1101/sqb.1981.045.01.019. [DOI] [PubMed] [Google Scholar]
  2. Biek D. P., Cohen S. N. Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF. J Bacteriol. 1989 Apr;171(4):2066–2074. doi: 10.1128/jb.171.4.2066-2074.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeLong A., Syvanen M. Membrane association of the Tnp and Inh proteins of IS50R. J Bacteriol. 1990 Sep;172(9):5516–5519. doi: 10.1128/jb.172.9.5516-5519.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Derbyshire K. M., Kramer M., Grindley N. D. Role of instability in the cis action of the insertion sequence IS903 transposase. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4048–4052. doi: 10.1073/pnas.87.11.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hopkins J. D., Clements M. B., Liang T. Y., Isberg R. R., Syvanen M. Recombination genes on the Escherichia coli sex factor specific for transposable elements. Proc Natl Acad Sci U S A. 1980 May;77(5):2814–2818. doi: 10.1073/pnas.77.5.2814. [DOI] [PMC free article] [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. Isberg R. R., Syvanen M. Replicon fusions promoted by the inverted repeats of Tn5. The right repeat is an insertion sequence. J Mol Biol. 1981 Jul 25;150(1):15–32. doi: 10.1016/0022-2836(81)90322-3. [DOI] [PubMed] [Google Scholar]
  9. 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]
  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. Kieny M. P., Lathe R., Lecocq J. P. New versatile cloning and sequencing vectors based on bacteriophage M13. Gene. 1983 Dec;26(1):91–99. doi: 10.1016/0378-1119(83)90039-2. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Krebs M. P., Reznikoff W. S. Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant. Gene. 1988 Mar 31;63(2):277–285. doi: 10.1016/0378-1119(88)90531-8. [DOI] [PubMed] [Google Scholar]
  14. Mazodier P., Cossart P., Giraud E., Gasser F. Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes. Nucleic Acids Res. 1985 Jan 11;13(1):195–205. doi: 10.1093/nar/13.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McCommas S. A., Syvanen M. Temporal control of transposition in Tn5. J Bacteriol. 1988 Feb;170(2):889–894. doi: 10.1128/jb.170.2.889-894.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Morisato D., Way J. C., Kim H. J., Kleckner N. Tn10 transposase acts preferentially on nearby transposon ends in vivo. Cell. 1983 Mar;32(3):799–807. doi: 10.1016/0092-8674(83)90066-1. [DOI] [PubMed] [Google Scholar]
  17. Rossetti O. L., Altman R., Young R. Kinetics of Tn5 transposition. Gene. 1984 Dec;32(1-2):91–98. doi: 10.1016/0378-1119(84)90036-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  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 Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES