Ac transposase binds in vitro to the Ac/Ds terminal inverted repeats
-- Heinz-Albert Becker and Reinhard Kunze

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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