Transposition of En/Spm requires a bi-functional protein

--Stefan Trentmann, Monika Frey, Heinz Saedler and Alfons Gierl

The En/Spm-encoded TNPA protein binds to 12bp sequence motifs in the subtermini of En/Spm (Gierl et al., EMBO J. 7:4045-4053). TNPA is required for excision of En/Spm and was proposed to serve as a "glue" for the association of the element's ends (Frey et al., EMBO J. 9:4037-4044). The formation of TNPA-DNA complexes was now analyzed in more detail by gel retardation, using individual binding motifs and in vitro translated TNPA protein.

Binding of TNPA to DNA leads to the formation of two complexes: a fast migrating complex that contains one TNPA molecule and a slower migrating complex that contains two TNPA molecules which are associated by protein-protein interaction. The deletion analysis of TNPA revealed two functional domains: one for DNA binding and one for dimerization of the protein. The region between position 122 to 427 constitutes the DNA binding domain, while amino acids 428 to 542 are required for the dimerization domain of TNPA. Mutant TNPA proteins that are defective in either the DNA binding or the dimerization domain are also defective with respect to promoting excision in the transgenic tobacco test system. We therefore propose that the association of the ends of En/Spm occurs via the dimerization domain of TNPA proteins that are bound to the various subterminal sites at each end of En/Spm.


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