Functional domains of the IS1 transposase: analysis in vivo and in vitro.
The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB' transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB', but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB' using a series of site-directed InsAB' mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding.
Ton-Hoang
Bao
B
Laboratoire de Microbiologie et de Génétique Moléculaire, CNRS, 118 route de Narbonne, 31062, Toulouse Cedex, France. tonhoang@ibcg.biotoul.fr
Turlan
Catherine
C
Chandler
Michael
M
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D019895:Transposases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016335:Zinc Fingers
D029968:Escherichia coli Proteins; C473497:IS1 transposase; C088046:InsB protein, E coli; D011506:Proteins; D012097:Repressor Proteins; C064548:insA1 protein, E coli; D019895:Transposases