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Figure 6 | Mobile DNA

Figure 6

From: Soluble expression, purification and characterization of the full length IS2 Transposase

Figure 6

Electrophoretic mobility shift assays. Binding efficiencies of the IS2 OrfAB Transposase derivatives from 22 randomly induced mutants. Reactions were carried out for 30 minutes at 20°C, with 10 nM of 32P-labeled annealed 50-mer oligonucleotides (except where stated in part (f) below) containing the inverted right repeat sequence and 0.11 μM of the partially purified mutant or wild type IS2 OrfAB-GFP protein derivatives (see Methods). Domain locations of the substitutions are color-coded and identified by a single letter code, that is, the binding domain (B) yellow, the leucine zipper-like (L) blue, the catalytic active site (C) green, and the middle interval (M) orange. Reactions were separated on 5% native polyacrylamide gels at 4°C at 120 mA as follows: (a) 450 Vhrs. (b) 420 Vhrs (c) 300 Vhrs (d) 450 Vhrs (e) 450 Vhrs (f) 12% native PAGE for 300 Vhrs using 87-mer annealed oligonucleotides. Binding efficiencies are identified as follows: 5 = Identical to that of the wild type, that is, absence of any dissociation of the complex. 4.5 = a slight loss of compactness of the undissociated complex seen in the wild type control. 4.0 = as in 4.5 but with a faster migrating tail of dissociated complexes. 3.5 = as in 4.0 but with a more prominent faster migrating tail of dissociated complexes. 3.0 = significant loss of compactness of the complex with a small amount of uncomplexed DNA. 2.5 = as in 3.0 but with significantly more uncomplexed DNA. 2.0 = as in 3.0 but mostly composed of uncomplexed DNA. 0.5 = mostly composed of uncomplexed DNA with a small tail of dissociated complex. 0 = no complex formation, identical to that of the protein-free controls (lane 1 in each panel) or the GFP control (part a lane 10). Double mutations are indicated within rectangular boxes. For GMF 18 the operative mutation, L97H, is shown in red (gel c, lane 4). GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hour

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