In an earlier publication we have reported that an Ac transposase (TPase) protein, overexpressed in insect cells, does not bind in vitro to the terminal inverted repeats (TIRs) of Ac/Ds (Kunze et al., EMBO J. 8:3177-3185, 1989). During the last year we have continued the DNA-binding studies with bacterially expressed and renatured TPase derivative lacking the amino-terminal 102 amino acids, TPase(103-807), and we investigated the influence of different incubation buffer compositions on the DNA-binding properties of the TPase. In addition, we could increase the sensitivity of the gel retardation assays by using a PhosphorImager for detection.
These modifications enabled us to detect a weak interaction of the TPase protein with the TIRs. This interaction is sequence-specific, as the mutated TIR sequence of the stable Ac18 element (Hehl and Baker, Mol. Gen. Genet. 217:53-59, 1989) is not recognized. The TPase/TIR-complexes have electrophoretic mobility similar to the complexes between TPase and the subterminal AAACGG motifs, i.e. they migrate as a diffuse band in agarose gels. However, the binding reactions differ: The TPase protein binds the subterminal AAACGG motifs with much higher affinity than the TIRs.
The Ac TIR sequence (C/TAGGGATGAAA) does not contain an A/TCG motif, which has been identified as the essential core sequence responsible for TPase binding to the subterminal AAACGG motifs (Becker and Kunze, MNL 68:21-22, 1994). We have therefore initiated experiments to determine whether the TPase has separate domains for binding to the TIRs and the subterminal motifs. We have obtained preliminary results indicating that the TPase-derivative TPase(103-465/R191H/H193R), which does not bind to the AAACGG motifs (Feldmar and Kunze, EMBO J. 10:4003-4010, 1991), has also lost the ability to bind to the TIRs. Accordingly, the basic TPase residues 191 and 193 are involved in recognition of the TIRs and the subterminal AAACGG motifs.
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