Activator encodes a transposase protein (TPase) which is crucially involved in the transposition reaction. TPase binds to repetitive subterminal sequence motifs and the terminal inverted repeats of Ac (Kunze and Starlinger, EMBO J. 8:3177-3185, 1989; Becker and Kunze, MNL 69:38, 1995). By immunochemical in situ staining it was found that the TPase forms large aggregates in the cell nuclei, and genetic experiments suggest that it acts as an oligomer (Heinlein et al., Plant J. 5:705-714; Kunze et al., PNAS 90:7094-7098, 1993). We assume that the TPase is the key and possibly sole protein component of a transposition complex (the "transpososome"), where it brings the ends of the transposable element and the new insertion site in close contact. In this model of the transpososome direct TPase interactions have a fundamental function. To localize the TPase protein/protein-interaction domain(s) we made use of the yeast two-hybrid-system. Initial experiments have demonstrated that the wild type TPase and a functional, amino-terminally truncated TPase(103-807) derivative, respectively, interact in the yeast cells (Essers and Kunze, MNL 69:41, 1995).

By progressive deletions from the amino- and carboxy-terminus of the TPase reading frame we have identified an approximately 100 amino acid domain close to the the carboxy-terminus (residues 664-754) which is required for a specific interaction with the TPase (103-807). A TPase derivative lacking 100 amino acids from the C-terminus [TPase (103-709)] does not interact with the full length TPase and the TPase (103-807), respectively. Thus, the TPase (664-754) domain is the only interaction domain detectable with the yeast two-hybrid-system.

The putative interaction domain contains a region (amino acids 685-750) which is highly conserved in transposase proteins of transposable elements originating from plant and insect species (Essers and Kunze, MNL 69:39-41, 1995). We have noticed earlier that an insertion of two amino acids within this conserved region at residue 709 results in complete inactivation of the protein in vivo, whereas similar insertions at the (non-conserved) residues 754 and 771 do not affect the transpositional activity (Kunze et al., PNAS 90:7094-7098, 1993). This correlates well with the results from the two-hybrid-system. The insertion at residue 709 abolishes protein/protein-interaction in yeast, whereas the 754 and 771 mutants still interact.

According to our experience it is important to verify the data obtained from the genetic two-hybrid-system by biochemical techniques. We have expressed the putative interaction domain (amino acids 674-777) with a N-terminal histidine-tag in E. coli and tested the fusion protein by chemical crosslinking experiments for protein-protein interaction. Preliminary results indicate that the fusion protein can be crosslinked with EGS [etyhlene glycol-bis(succinic acid N-hydroxysuccinimide ester)] at standard concentrations, whereas no crosslinking of the control protein lysozyme was observed.

As the self-interacting TPase protein fragment consists of approximately 100 amino acids, it is likely that it contains only one interaction domain. It probably mediates a symmetric interaction ("head-to-head") between two TPase monomers. The TPase binds to the subterminal regions of Ac and Ds elements at multiple, five or six bp target sites which are frequently arranged as direct repeats. Thus, it seems unlikely that the proposed "head-to-head" contacts are involved in stabilization of neighbouring TPase molecules on one end of the transposon. However, such contacts could mediate the conjunction between the two transposable element ends in a transpososome. The tight connection between both Ac ends may be a prerequisite for the initiation of the excision reaction. 

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