Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Oct;179(20):6238–6243. doi: 10.1128/jb.179.20.6238-6243.1997

Functional domains of the InsA protein of IS2.

G S Lei 1, S T Hu 1
PMCID: PMC179535  PMID: 9335268

Abstract

The InsA protein is a transcriptional regulator. It binds to the promoter region of insA and insAB'. To understand the molecular mechanism for the interaction between InsA and its binding sequence, the functional domains of InsA were identified. The glutaraldehyde cross-linking method and the two-hybrid expression system were used to study the protein-protein interaction of InsA. The results of these experiments showed that InsA forms homodimers. Deletion of the last 44 amino acid residues at its C terminus, but not the first 12 or 57 residues at the N terminus, abolished the ability of InsA to form homodimers, indicating that the protein-protein interaction domain of InsA is located at its C terminus. Gel retardation assays revealed that deletion of the last 29 amino acid residues at its C terminus had no effect on the DNA binding ability of InsA. In contrast, deletion of the first N-terminal 12 residues abolished the DNA binding capability of InsA. These results indicate that the DNA binding domain of InsA is located at its N terminus.

Full Text

The Full Text of this article is available as a PDF (1,005.7 KB).

Selected References

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

  1. Berger B., Wilson D. B., Wolf E., Tonchev T., Milla M., Kim P. S. Predicting coiled coils by use of pairwise residue correlations. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8259–8263. doi: 10.1073/pnas.92.18.8259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Crewther W. G., Inglis A. S., McKern N. M. Amino acid sequences of alpha-helical segments from S-carboxymethylkerateine-A. Complete sequence of a type-II segment. Biochem J. 1978 Aug 1;173(2):365–371. doi: 10.1042/bj1730365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fiandt M., Szybalski W., Malamy M. H. Polar mutations in lac, gal and phage lambda consist of a few IS-DNA sequences inserted with either orientation. Mol Gen Genet. 1972;119(3):223–231. doi: 10.1007/BF00333860. [DOI] [PubMed] [Google Scholar]
  5. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gough K. H., Inglis A. S., Crewther W. G. Amino acid sequences of alpha-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment. Biochem J. 1978 Aug 1;173(2):373–385. doi: 10.1042/bj1730373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hirsch H. J., Starlinger P., Brachet P. Two kinds of insertions in bacterial genes. Mol Gen Genet. 1972;119(3):191–206. doi: 10.1007/BF00333858. [DOI] [PubMed] [Google Scholar]
  8. Hu S. T., Hwang J. H., Lee L. C., Lee C. H., Li P. L., Hsieh Y. C. Functional analysis of the 14 kDa protein of insertion sequence 2. J Mol Biol. 1994 Feb 18;236(2):503–513. doi: 10.1006/jmbi.1994.1161. [DOI] [PubMed] [Google Scholar]
  9. Hu S. T., Lee C. H. Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli. Mol Gen Genet. 1988 Nov;214(3):490–495. doi: 10.1007/BF00330485. [DOI] [PubMed] [Google Scholar]
  10. Hu S. T., Lee L. C., Lei G. S. Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2. J Bacteriol. 1996 Oct;178(19):5652–5659. doi: 10.1128/jb.178.19.5652-5659.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Iwabuchi K., Li B., Bartel P., Fields S. Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene. 1993 Jun;8(6):1693–1696. [PubMed] [Google Scholar]
  12. Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  15. Lee C. H., Hu S. T., Swiatek P. J., Moseley S. L., Allen S. D., So M. Isolation of a novel transposon which carries the Escherichia coli enterotoxin STII gene. J Bacteriol. 1985 May;162(2):615–620. doi: 10.1128/jb.162.2.615-620.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Li B., Fields S. Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system. FASEB J. 1993 Jul;7(10):957–963. doi: 10.1096/fasebj.7.10.8344494. [DOI] [PubMed] [Google Scholar]
  17. Mahillon J., Seurinck J., van Rompuy L., Delcour J., Zabeau M. Nucleotide sequence and structural organization of an insertion sequence element (IS231) from Bacillus thuringiensis strain berliner 1715. EMBO J. 1985 Dec 30;4(13B):3895–3899. doi: 10.1002/j.1460-2075.1985.tb04163.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nisen P., Purucker M., Shapiro L. Deoxyribonucleic acid sequence homologies among bacterial insertion sequence elements and genomes of various organisms. J Bacteriol. 1979 Nov;140(2):588–596. doi: 10.1128/jb.140.2.588-596.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  20. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  21. Rezsöhazy R., Hallet B., Delcour J., Mahillon J. The IS4 family of insertion sequences: evidence for a conserved transposase motif. Mol Microbiol. 1993 Sep;9(6):1283–1295. doi: 10.1111/j.1365-2958.1993.tb01258.x. [DOI] [PubMed] [Google Scholar]
  22. Rezsöhazy R., Hallet B., Mahillon J., Delcour J. IS231V and W from Bacillus thuringiensis subsp. israelensis, two distant members of the IS231 family of insertion sequences. Plasmid. 1993 Sep;30(2):141–149. doi: 10.1006/plas.1993.1041. [DOI] [PubMed] [Google Scholar]
  23. Saedler H., Heiss B. Multiple copies of the insertion-DNA sequences IS1 and IS2 in the chromosome of E. coli K-12. Mol Gen Genet. 1973 May 9;122(3):267–277. doi: 10.1007/BF00278602. [DOI] [PubMed] [Google Scholar]
  24. Sodek J., Hodges R. S., Smillie L. B., Jurasek L. Amino-acid sequence of rabbit skeletal tropomyosin and its coiled-coil structure. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3800–3804. doi: 10.1073/pnas.69.12.3800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stone D., Smillie L. B. The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence. J Biol Chem. 1978 Feb 25;253(4):1137–1148. [PubMed] [Google Scholar]
  26. 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]

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

RESOURCES